Von Willebrand factor-and factor VIII-polymer conjugates having a releasable linkage

ABSTRACT

The present invention provides von Willebrand Factor-polymer conjugates and Factor VIII-polymer conjugates, each having a releasable linkage. Methods of making conjugates, methods for administering conjugates, are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of priority to U.S. provisionalapplication Ser. No. 60/877,531, filed Dec. 27, 2006, which isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to polymer-active agentconjugates having a releasable linkage to thereby release the activeagent in vivo. In addition, the invention relates to, among otherthings, methods for synthesizing the conjugates, methods for purifyingthe conjugates, and so on.

BACKGROUND OF THE INVENTION

Scientists and clinicians face a number of challenges in their attemptsto develop active agents into forms suited for delivery to a patient.Active agents that are polypeptides, for example, are often deliveredvia injection rather than orally. In this way, the polypeptide isintroduced into the systemic circulation without exposure to theproteolytic environment of the stomach. Injection of polypeptides,however, has several drawbacks. For example, many polypeptides have arelatively short half-life, thereby necessitating repeated injections,which are often inconvenient and painful. Moreover, some polypeptidescan elicit one or more immune responses with the consequence that thepatient's immune system attempts to destroy or otherwise neutralize theimmunogenic polypeptide. Of course, once the polypeptide has beendestroyed or otherwise neutralized, the polypeptide cannot exert itsintended pharmacodynamic activity. Thus, delivery of active agents suchas polypeptides is often problematic even when these agents areadministered by injection.

Some success has been achieved in addressing the problems of deliveringactive agents via injection. For example, conjugating the active agentto a water-soluble polymer has resulted in a polymer-active agentconjugate having reduced immunogenicity and antigenicity. In addition,these polymer-active agent conjugates often have greatly increasedhalf-lives compared to their unconjugated counterparts as a result ofdecreased clearance through the kidney and/or decreased enzymaticdegradation in the systemic circulation. As a result of having a greaterhalf-life, the polymer-active agent conjugate requires less frequentdosing, which in turn reduces the overall number of painful injectionsand inconvenient visits with a health care professional. Moreover,active agents that were only marginally soluble demonstrate asignificant increase in water solubility when conjugated to awater-soluble polymer.

Due to its documented safety as well as its approval by the FDA for bothtopical and internal use, polyethylene glycol has been conjugated toactive agents. When an active agent is conjugated to a polymer ofpolyethylene glycol or “PEG,” the conjugated active agent isconventionally referred to as “PEGylated.” The commercial success ofPEGylated active agents such as PEGASYS® PEGylated interferon alpha-2a(Hoffmann-La Roche, Nutley, N.J.), PEG-INTRON® PEGylated interferonalpha-2b (Schering Corp., Kennilworth, N.J.), and NEULASTA™PEG-filgrastim (Amgen Inc., Thousand Oaks, Calif.) demonstrates thatadministration of a conjugated form of an active agent can havesignificant advantages over the unconjugated counterpart. Smallmolecules such as distearoylphosphatidylethanolamine (Zalipsky (1993)Bioconjug. Chem. 4(4):296-299) and fluorouracil (Ouchi et al. (1992)Drug Des. Discov. 9(1):93-105) have also been PEGylated. Harris et al.have provided a review of the effects of PEGylation on pharmaceuticals.Harris et al. (2003) Nat. Rev. Drug Discov. 2(3):214-221.

Despite these successes, conjugation of a polymer to an active agent toresult in a commercially relevant drug is often challenging. Forexample, conjugation can result in the polymer being attached at or neara site on the active agent that is necessary for pharmacologic activity(e.g., at or near a binding site). Such conjugates may therefore haveunacceptably low activity due to, for example, the steric effectsintroduced by the polymer. Attempts to remedy conjugates havingunacceptably low activity can be frustrated when the active agent hasfew or no other sites suited for attachment to a polymer. Thus,additional PEGylation alternatives have been desired.

One suggested approach for solving this and other problems is“reversible PEGylation” wherein the native active agent (or a moietyhaving increased activity compared to the PEGylated active agent) isreleased. For example, reversible PEGylation has been disclosed in thefield of cancer chemotherapies. See Greenwald (1997) Exp. Opin. Ther.Patents 7(6):601-609. U.S. Patent Application Publication No.2005/0079155 describes conjugates using reversible linkages. Asdescribed in this publication, reversible linkages can be effectedthrough the use of an enzyme substrate moiety. It has been pointed out,however, that approaches relying on enzymatic activity are dependent onthe availability of enzymes. See Peleg-Schulman (2004) J. Med. Chem.47:4897-4904. Patient variability around the amount and activity ofthese enzymes can introduce inconsistent performance of the conjugateamong different populations. Thus, additional approaches that do notrely on enzymatic processes for polymer release have been described asbeing desirable.

Another approach for reversible PEGylation is described in U.S. Pat. No.7,060,259, which described (among other things) water-soluble prodrugsin which a biologically active agent is linked to a water-solublenon-immunogenic polymer by a hydrolyzable carbamate bond. As describedtherein, the biologically active agent can be readily released by thehydrolysis of the carbmate bond in vivo without the need for addingenzymes or catalytic materials.

Another approach for reversible PEGylation is described inPeleg-Schulman (2004) J. Med. Chem. 47:4897-4904, WO 2004/089280 andU.S. Patent Application Publication No. 2006/0171920. Although thisapproach has been applied to a limited number of active agents, thesereferences ignore other active agents for which reversible PEGylationwould be particularly suited. Yet another releaseable approach isdescribed in U.S. Patent Application Publication No. 2006/0293499.

In the area of bleeding disorders, proteins (such as, for example, vonWillebrand Factor and Factor VIII) can sometimes be administered to apatient to address or otherwise ameliorate the bleeding disorder. Due tothe relatively short half-life of such proteins, it would beadvantageous to increase the in vivo half-life of these proteins by, forexample, reversible PEGylation. Thus, the present invention seeks tosolve this and other needs in the art.

SUMMARY OF THE INVENTION

In one or more embodiments of the invention, a conjugate of thefollowing formula is provided:

wherein:

POLY¹ is a first water-soluble polymer;

POLY² is a second water-soluble polymer;

X¹ is a first spacer moiety;

X² is a second spacer moiety;

H_(α) is an ionizable hydrogen atom;

R¹ is H or an organic radical;

R² is H or an organic radical;

(a) is either zero or one;

(b) is either zero or one;

R^(e1), when present, is a first electron altering group;

R^(e2), when present, is a second electron altering group; and

Y¹ is O or S;

Y² is O or S; and

(vWF/F8) is a residue of an amine-containing biologically active agentselected from the group consisting of a von Willebrand Factor moiety anda Factor VIII moiety.

In one or more embodiments of the invention, methods for preparingconjugates are provided.

In one or more embodiments of the invention, pharmaceutical preparationscomprising the conjugates are provided.

In one or more embodiments of the invention, methods for administeringthe conjugates are provided.

In one or more embodiments of the invention, a construct is provided,the construct comprising a conjugate as provided herein bound to atleast one Factor VIII moiety.

In one or more embodiments of the invention, a von WillebrandFactor-water soluble polymer conjugate is provided, the conjugate havingan in vivo half-life increased by a factor of at least 1.5 as comparedto the in vivo half-life of a von Willebrand Factor moiety notconjugated to the water-soluble polymer.

In one or more embodiments of the invention, a von WillebrandFactor-water soluble polymer conjugate is provided, the conjugate havingan in vivo half-life increased by a factor of at least 2 as compared tothe in vivo half-life of a von Willebrand Factor moiety not conjugatedto the water-soluble polymer.

In one or more embodiments of the invention, a Factor VIII moiety-watersoluble polymer conjugate is provided, the conjugate having an in vivohalf-life increased by a factor of at least 1.5 as compared to the invivo half-life of a Factor VIII moiety not conjugated to thewater-soluble polymer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a typical chromatogram of a conjugate composition preparedin accordance with the procedure set forth in Example 1A.

FIG. 2A shows a typical separation profile of a conjugate compositionprepared in accordance with the procedure set forth in Example 2A.

FIG. 2B shows a typical chromatogram of a conjugate composition preparedin accordance with the procedure set forth in Example 2A.

FIG. 3A shows a chromatogram following anion exchange chromatography ofa conjugate composition prepared in accordance with the procedure setforth in Example 3A.

FIG. 3B shows a gel following SDS-PAGE analysis under reduced conditionsof a conjugate composition prepared in accordance with the procedure setforth in Example 3A. NuPAGE Novex Tris-Acetate Gel (3-8%) withTris-Acetate SDS Running Buffer. The gel was stained by Pierce GelCodeBlue stain. Lane 1: Invitrogen HiMark Unstained High Molecular WeightProtein Standard. Lane 2: rVWF standard. Lane 3: conjugate.

FIG. 3C shows a gel following SDS-PAGE analysis under non-reducedconditions of a conjugate composition prepared in accordance with theprocedure set forth in Example 3A. NuPAGE Novex Tris-Acetate Gel (3-8%)with Tris-Acetate SDS Running Buffer. The PEG was detected by Bariumchloride/iodine stain. Lane 1: conjugate. Lane 2: 0.002 wt/v % of PEG20Kcontrol. Lane 3: 0.005 wt/v % of PEG20K control. Lane 4: 0.01 wt/v % ofPEG20K control.

FIGS. 4A and 4B show chromatograms following ion exchange chromatographyof conjugate compositions prepared in accordance with the procedures setforth in Examples 3B and 3C, respectively.

FIG. 5 shows a chromatogram of a conjugate composition prepared inaccordance with the procedure set forth in Example 4A.

FIGS. 6A and 6B show a gels following SDS-PAGE analysis using BariumIodided staining and Coomassie staining, respectively, of a conjugatecomposition prepared in accordance with the procedures set forth inExample 4A.

FIG. 7 shows the structural characterization of native rVWF 133P1 byelectrophoresis. Panel A: Reduced SDS-PAGE followed by silver-staining.Panel B: Reduced SDS-PAGE followed by Coomassie staining. Panel C:Immunoblot with a polyclonal anti-human VWF antibody of the gels of thereduced SDS-PAGE. Panel D: VWF multimer distribution visualized by 2.5%agarose gel electrophoresis detected with anti VWF antibody. Furtherinformation concerning this figure is provided in Example 5.

FIG. 8 shows the domain structure of releasable PEGylated rVWFconjugates visualized by reducing SDSPAGE with protein staining. PanelA: Reduced SDS-PAGE followed by silver-staining. Panel B: ReducedSDS-PAGE followed by Coomassie staining. Further information concerningthis figure is provided in Example 5.

FIG. 9 shows the domain structure of releasable PEGylated rVWFvisualized by immunoblots of reducing SDS-PAGE specific for VWF and PEG.Panel A: Immunoblot with a polyclonal anti-human VWF antibody of thegels of the reduced SDS-PAGE. Panel B: Immunoblot with a polyclonalanti-PEG antibody of the gels of the reduced SDS-PAGE. Furtherinformation concerning this figure is provided in Example 5.

FIG. 10 shows the VWF multimer distribution of releasable rVWFconjugates visualized by low resolution agarose gel electrophoresis.Panel A: Multimer distribution detected with anti VWF antibody in thegel. Panel B: PEGylated VWF multimers detected with anti PEG antibodyafter immunolotting. Further information concerning this figure isprovided in Example 5.

FIG. 11 shows the fine structure of VWF multimers of the releasable rVWFconjugates visualized by high-resolution agarose gel electrophoresis.Panel A: VWF multimer structure visualized by anti VWF antibody in thegel. Panel B: PEGylated VWF multimers detected with anti PEG antibodyafter immunolotting. Further information concerning this figure isprovided in Example 5.

FIG. 12 shows the FVIII-binding capacity of the releasable PEG-rVWFconjugates in the presence of unmodified rVWF under flow conditions.Squares, open: PEGylated rVWF Lys 20K br rel short and rFVIII; squares:PEGylated rVWF Lys 20K br rel long and and rFVIII; triangles, open:PEGylated rVWF Lys 40K br rel short and rFVIII; triangles: PEGylatedrVWF Lys 40K br rel long and rFVIII; circles, open: PEGylated rVWF Lys60K br rel short and rFVIII; circles: PEGylated rVWF Lys 60K br rel longand rFVIII; star: native rVWF (133 pool1) and rFVIII; cross: rproVWF198and rFVIII. Further information concerning this figure is provided inExample 5.

FIG. 13 shows the changes in VWF:CB activity of VWF in the ADAMTS13digested samples. Squares, open: PEGylated rVWF Lys 20K br rel short;squares: PEGylated rVWF Lys 20K br rel long; triangles, open: PEGylatedrVWF Lys 40K br rel short; triangles: PEGylated rVWF Lys 40K br rellong; star: native rVWF (133 P1). Further information concerning thisfigure is provided in Example 5.

FIG. 14 shows the ADAMTS 13-mediated satellite band formation in rVWFvisualized by SDS-agarose gel. Further information concerning thisfigure is provided in Example 5.

FIG. 15 shows the time course of changes in PEGylation degreedemonstrated by anti-PEG immunoblot. Further information concerning thisfigure is provided in Example 5.

FIG. 16 shows the comparison of the native rVWF and PEGylated rVWF Lys20K br short low (both with co-injected rFVIII) in FVIII-deficientknockout mice. A: Time-dependent changes in VWF:Ag. B: Time-dependentchanges in FVIII:activity. Data are displayed as IU VWF:Ag/ml or IUFVIII/ml mouse plasma. Circles: PEGylated rVWF (1.6 mg/kg) andrFVIII.(200 IU/kg); triangles: native rVWF (1.6 mg/kg) and rFVIII (200IU/kg). The symbols show the mean values±SD of the 6 plasma samplesobtained at each time point. Further information concerning this figureis provided in Example 5.

FIG. 17 shows the comparison of the native rVWF and PEGylated rVWF Lys20K br long low (both with co-injected rFVIII) in FVIII-deficientknockout mice. A: Time-dependent changes in VWF:Ag. B: Time-dependentchanges in FVIII:activity. Data are displayed as IU VWF:Ag/ml or IUFVIII/ml mouse plasma. Circles: PEGylated rVWF (1.6 mg/kg) and rFVIII(200 IU/kg); triangles: native rVWF (1.6 mg/kg) and rFVIII (200 IU/kg).The symbols show the mean values±SD of the 6 plasma samples obtained ateach time point. Further information concerning this figure is providedin Example 5.

FIG. 18 shows the comparison of the native rVWF and PEGylated rVWF Lys40K br short low (both with coinjected rFVIII) in FVIII-deficientknockout mice. A: Time-dependent changes in VWF:Ag. B: Time-dependentchanges in FVIII:activity. Data are displayed as IU VWF:Ag/ml or IUFVIII/ml mouse plasma. Circles: PEGylated rVWF (1.6 mg/kg) and rFVIII(180 IU/kg); triangles: native rVWF (1.6 mg/kg) and rFVIII (190 IU/kg).The symbols show the mean values±SD of the 6 plasma samples obtained ateach time point. Further information concerning this figure is providedin Example 5.

FIG. 19 shows the comparison of the native rVWF and PEGylated rVWF Lys40K br long low (both with co-injected rFVIII) in FVIII-deficientknockout mice. A: Time-dependent changes in VWF:Ag. B: Time-dependentchanges in FVIII:activity. Data are displayed as IU VWF:Ag/ml or IUFVIII/ml mouse plasma. Circles: PEGylated rVWF (1.6 mg/kg) and rFVIII(190 IU/kg); triangles: native rVWF (1.6 mg/kg) and rFVIII (190 IU/kg).The symbols show the mean values±SD of the 6 plasma samples obtained ateach time point. Further information concerning this figure is providedin Example 5.

FIG. 20 shows the comparison of the native rVWF and PEGylated rVWF Lys60K br short low (both with co-injected rFVIII) in FVIII-deficientknockout mice. A: Time-dependent changes in VWF:Ag. B: Time-dependentchanges in FVIII:activity. Data are displayed as IU VWF:Ag/ml or IUFVIII/ml mouse plasma. Circles: PEGylated rVWF (1.6 mg/kg) and rFVIII(200 IU/kg); triangles: native rVWF (1.6 mg/kg) and rFVIII (200 IU/kg).The symbols show the mean values±SD of the 6 plasma samples obtained ateach time point. Further information concerning this figure is providedin Example 5.

FIG. 21 shows the comparison of the native rVWF and PEGylated rVWF Lys60K br long low (both with co-injected rFVIII) in FVIII-deficientknockout mice. A: Time-dependent changes in VWF:Ag. B: Time-dependentchanges in FVIII:activity. Data are displayed as IU VWF:Ag/ml or IUFVIII/ml mouse plasma. Circles: PEGylated rVWF (1.6 mg/kg) and rFVIII(200 IU/kg); triangles: native rVWF (1.6 mg/kg) and rFVIII (200 IU/kg).The symbols show the mean values±SD of the 6 plasma samples obtained ateach time point. Further information concerning this figure is providedin Example 5.

FIG. 22 shows the PEGylated rVWF candidates summary. A: Time-dependentchanges in VWF:Ag. B: Time-dependent changes in FVIII:activity. Opensquares: PEGylated rVWF Lys 20K br short low and rFVIII; squares:PEGylated rVWF Lys 20K br long low and rFVIII; open triangels: PEGylatedrVWF Lys 40K br short low and rFVIII; triangles: PEGylated rVWF Lys 40Kbr long low and rFVIII; circles: PEGylated rVWF Lys 60K br short low andrFVIII; circles: PEGylated rVWF Lys 60K br long low and rFVIII; andstars: native rVWF (133 pool1) and rFVIII. Further informationconcerning this figure is provided in Example 5.

FIG. 23 shows the dose-adjusted AUC for VWF:Ag. Further informationconcerning this figure is provided in Example 5.

FIG. 24 shows the AUC and half life for FVIII, co-injected withPEGylated rVWF candidates. Further information concerning this figure isprovided in Example 5.

FIG. 25 shows the MRT for FVIII, co-injected with PEGylated rVWFcandidates. Further information concerning this figure is provided inExample 5.

FIG. 26 shows the domain structure of the native rFVIII (MOQ HEPES 01-E)visualized by reducing SDS-PAGE followed by immunoblot with a polyclonalanti-human FVIII antibody. Further information concerning this figure isprovided in Example 6.

FIG. 27 shows the quantitative parameters of the FXa-generation curve.Further information concerning this figure is provided in Example 6.

FIG. 28 shows the domain structure of releasable PEG-rFVIII conjugatesvisualized by reducing SDSPAGE followed by immunoblot. Panel A:Immunoblot with a polyclonal anti-human FVIII antibody. Panel B:Immunoblot with a polyclonal antibody directed against PEG. Furtherinformation concerning this figure is provided in Example 6.

FIG. 29 shows the structure of HC and LC of releasable PEG-rFVIIIconjugates visualized by reducing SDS-PAGE followed by immunoblots.Panel A: Immunoblot with a monoclonal anti-human FVIII HC-A2 domainantibody. Panel B: Immunoblot with a monoclonal anti-human FVIII LC-A3domain antibody. Further information concerning this figure is providedin Example 6.

FIG. 30 shows the Factor Xa (“FXa”) generation curves in presence ofnon-activated PEG-rFVIII. Stars: native rFVIII MOQ HEPES 01-E; circles:PEG-rFVIII Lys 20K br short; triangles: PEG-rFVIII Lys 40K br short;squares: PEG-rFVIII Lys 60K br short; cross: FVIII control; circles,open: PEG-rFVIII Lys 20K br long; triangles, open: PEG-rFVIII Lys 40K brlong; squares, open: PEG-rFVIII Lys 60K br long. Further informationconcerning this figure is provided in Example 6.

FIG. 31 shows the FXa generation curves in presence ofthrombin-activated PEG-rFVIII. Stars: native rFVIII MOQ HEPES 01-E;circles: PEG-rFVIII Lys 20K br short; triangles: PEG-rFVIII Lys 40K brshort; squares: PEG-rFVIII Lys 60K br short; cross: FVIII control;circles, open: PEG-rFVIII Lys 20K br long; triangles, open: PEG-rFVIIILys 40K br long; squares, open: PEG-rFVIII Lys 60K br long. Furtherinformation concerning this figure is provided in Example 6.

FIG. 32 shows the activation and inactivation of PEG-rFVIII by thrombin.Stars: native rFVIII MOQ HEPES 01-E; circles: PEG-rFVIII Lys 20K brshort; triangles: PEG-rFVIII Lys 40K br short; squares: PEG-rFVIII Lys60K br short; cross: FVIII control; circles, open: PEG-rFVIII Lys 20K brlong; triangles, open: PEG-rFVIII Lys 40K br long; squares, open:PEG-rFVIII Lys 60K br long. Further information concerning this figureis provided in Example 6.

FIG. 33 shows the APC-mediated inactivation of PEGylated FVIIIconjugates. Stars: native rFVIII MOQ HEPES 01-E; circles: PEG-rFVIII Lys20K br short; triangles: PEG-rFVIII Lys 40K br short; squares:PEG-rFVIII Lys 60K br short; cross: FVIII control; circles, open:PEG-rFVIII Lys 20K br long; triangles, open: PEG-rFVIII Lys 40K br long;squares, open: PEG-rFVIII Lys 60K br long. Further informationconcerning this figure is provided in Example 6.

FIG. 34 shows the APC-mediated inactivation of thrombin activatedPEGylated FVIII conjugates. Stars: native rFVIII MOQ HEPES 01-E;circles: PEG-rFVIII Lys 20K br short; triangles: PEG-rFVIII Lys 40K brshort; squares: PEG-rFVIII Lys 60K br short; cross: FVIII control;circles, open: PEG-rFVIII Lys 20K br long; triangles, open: PEG-rFVIIILys 40K br long; squares, open: PEG-rFVIII Lys 60K br long. Furtherinformation concerning this figure is provided in Example 6.

FIG. 35 shows the improvement of thrombin generation of aFVIII-deficient plasma by in vitro addition of native rFVIII. Panel A:Thrombin generation curves obtained with rFVIII MOQ HEPES 01-E spikedinto FVIII deficient plasma; line a: without rFVIII; line b: 0.0025 μgrFVIII/ml; line c: 0.01 μg rFVIII/ml; line d: 0.025 μg rFVIII/ml; linee: 0.1 μg rFVIII/ml. Panel B: Linear dose response curves of nativerFVIII MOQ HEPES 01-E. Further information concerning this figure isprovided in Example 6.

FIG. 36 shows the thrombin generation curves (Panels A-F) obtained withthe PEG-rFVIII samples in the FVIII-deficient plasma and thedose-response curves (Panel G) of the peak thrombin values. Stars:native rFVIII MOQ HEPES 01-E; circles: PEG-rFVIII Lys 20K br short;triangles: PEG-rFVIII Lys 40K br short; squares: PEG-rFVIII Lys 60K brshort; cross: FVIII control; circles, open: PEG-rFVIII Lys 20K br long;triangles, open: PEG-rFVIII Lys 40K br long; squares, open: PEG-rFVIIILys 60K br long. Further information concerning this figure is providedin Example 6.

FIG. 37 shows the recovery of in FVIII-specific activity upon incubationin buffer at pH 8.1. Panel A: native rFVIII MOQ HEPES 01-E (stars) andFVIII control (cross). Panel B: PEG-rFVIII Lys 20K br short (closedcircles) and long (open circles). Panel C: PEG-rFVIII Lys 40K br short(closed triangles) and long (open triangles). Panel D: PEG-rFVIII Lys60K br short (closed squares) and long (open squares). Furtherinformation concerning this figure is provided in Example 6.

FIG. 38 shows the recovery of FVIII:Ag upon incubation in buffer at pH8.1. Panel A: native rFVIII MOQ HEPES 01-E (stars) and FVIII control(cross). Panel B: PEG-rFVIII Lys 20K br short (closed circles) and long(open circles). Panel C: PEG-rFVIII Lys 40K br short (closed triangles)and long (open triangles). Panel D: PEG-rFVIII Lys 60K br short (closedsquares) and long (open squares). Further information concerning thisfigure is provided in Example 6.

FIG. 39 shows the structural changes of FVIII upon incubation atincreased pH demonstrated by anti-FVIII immunoblot. Further informationconcerning this figure is provided in Example 6.

FIG. 40 shows the structural changes of FVIII upon incubation atincreased pH demonstrated by anti-FVIII HC-A2 domain immunoblot. Furtherinformation concerning this figure is provided in Example 6.

FIG. 41 shows the structural changes of FVIII upon incubation atincreased pH demonstrated by anti-PEG immunoblot. Further informationconcerning this figure is provided in Example 6.

FIG. 42 shows the changes in FVIII-specific activities of the PEG-rFVIIIupon incubation in FVIII-deficient plasma at +37° C. Panel A: Changes inFVIII activity upon incubation expressed as IU FVIII:Chrom activity/mgprotein. Panel B: Changes of the FVIII specific activities relative tothe initial value expressed as % of the initial values measuredimmediately after the addition to the plasma. Symbols: black stars,native rFVIII MOQ HEPES 01-E; closed circles, PEG-rFVIII Lys 20K brshort; open circles, PEG-rFVIII Lys 20K br long; closed triangles,PEG-rFVIII Lys 40K br short; open triangles, PEG-rFVIII Lys 40K br long.Further information concerning this figure is provided in Example 6.

FIG. 43 shows the changes in the FVIII antigen to protein ratio of thePEG-rFVIII upon incubation in FVIII-deficient plasma at +37° C. Panel A:Changes in ratio of FVIII antigen/protein upon incubation expressed asIU FVIII:Ag/mg protein. Panel B: Changes of the ratio FVIIIantigen/protein relative to the initial value expressed as % of theinitial values measured immediately after the addition to the plasma.Symbols: black stars, native rFVIII MOQ HEPES 01-E; closed circles,PEG-rFVIII Lys 20K br short; open circles, PEG-rFVIII Lys 20K br long;closed triangles, PEG-rFVIII Lys 40K br short; open triangles:PEG-rFVIII Lys 40K br long. Further information concerning this figureis provided in Example 6.

FIG. 44 shows the comparison of native rFVIII and PEG-rFVIII Lys 20K brshort in FVIII-deficient knockout mice. Panel A: absolute FVIII activitylevels in plasma; closed circles, PEG-rFVIII (320 IU/kg, 168 μg/kg);closed triangles, native rFVIII (170 IU/kg, 25 μg/kg). The symbols showthe mean values±SD of the 6 plasma samples obtained at each point oftime. Further information concerning this figure is provided in Example6.

FIG. 45 shows the comparison of native rFVIII and PEG-rFVIII Lys 20K brlong in FVIII-deficient knockout mice. Panel A: absolute FVIII activitylevels in plasma; closed circles, PEG-rFVIII (210 IU/kg, 164 μg/kg);closed triangles, native rFVIII (200 IU/kg, 35 μg/kg). The symbols showthe mean values±SD of the 6 plasma samples obtained at each point oftime. Further information concerning this figure is provided in Example6.

FIG. 46 shows the comparison of native rFVIII and PEG-rFVIII Lys 40K brshort in FVIII-deficient knockout mice. Panel A: absolute FVIII activitylevels in plasma; closed circles, PEG-rFVIII (230 IU/kg, 94 μg/kg);closed triangles: native rFVIII (230 IU/kg, 32 μg/kg). The symbols showthe mean values±SD of the 6 plasma samples obtained at each point oftime. Further information concerning this figure is provided in Example6.

FIG. 47 shows the comparison of native rFVIII and PEG-rFVIII Lys 40K brlong in FVIII-deficient knockout mice. Panel A: absolute FVIII activitylevels in plasma; closed circles: PEG-rFVIII (230 IU/kg, 94 μg/kg);closed triangles: native rFVIII (230 IU/kg, 32 μg/kg). The symbols showthe mean values±SD of the 6 plasma samples obtained at each point oftime. Further information concerning this figure is provided in Example6.

FIG. 48 shows the comparison of native rFVIII and PEG-rFVIII Lys 60K brshort in FVIII-deficient knockout mice. Panel A: absolute FVIII activitylevels in plasma; closed circles, PEG-rFVIII (200 IU/kg, 133 μg/kg);closed triangles: native rFVIII (190 IU/kg, 32 μg/kg). The symbols showthe mean values±SD of the 6 plasma samples obtained at each point oftime. Further information concerning this figure is provided in Example6.

FIG. 49 shows the comparison of native rFVIII and PEG-rFVIII Lys 60K brlong in FVIII-deficient knockout mice. Panel A: absolute FVIII activitylevels in plasma; closed circles: PEG-rFVIII (170 IU/kg, 62 μg/kg);closed triangles: native rFVIII (190 IU/kg, 32 μg/kg). The symbols showthe mean values±SD of the 6 plasma samples obtained at each point oftime. Further information concerning this figure is provided in Example6.

FIG. 50 shows the comparison of native rFVIII and PEGylated rFVIIIconjugates in FVIII deficient mice. Closed circles, PEG rFVIII Lys 20Kbr short; open circles, PEG rFVIII Lys 20K br long; closed triangles,PEG rFVIII Lys 40K br short; open triangles, PEG rFVIII Lys 40K br long;closed squares, PEG rFVIII Lys 60K br short; open squares, PEG rFVIIILys 60K br long; open diamonds, native rFVIII. The symbols show the“normalized %” mean values±SD of the 6 plasma samples obtained at eachtime point (for PEG-rFVIII) or the mean values±SD of the 24 plasmasamples obtained at each time point (native rFVIII). Further informationconcerning this figure is provided in Example 6.

FIG. 51 shows the dose-adjusted AUC and half-life for native rFVIII andPEG-rFVIII conjugates. Panel A: Area under the curve (dose adjusted).The symbols show the mean values±95% confidence intervals for therespective PEG-rFVIII conjugate; data for rFVIII native are the mean±95%confidence intervals of all control groups performed, equivalent to 24animals per time point; open squares: native rFVIII; closed squares:PEG-rFVIII. Further information concerning this figure is provided inExample 6.

FIG. 52 shows the mean residence time (“MRT”) for native rFVIII andPEG-rFVIII conjugates. Mean residence time and range for rFVIII control(open square, mean of all control groups, 24 animal per sampling point)and for PEG-rFVIII candidates (closed squares). Further informationconcerning this figure is provided in Example 6.

DETAILED DESCRIPTION OF THE INVENTION

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular polymers,synthetic techniques, active agents, and the like, as such may vary.

It must be noted that, as used in this specification and the claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to a“polymer” includes a single polymer as well as two or more of the sameor different polymers, reference to a “conjugate” refers to a singleconjugate as well as two or more of the same or different conjugates,reference to an “excipient” includes a single excipient as well as twoor more of the same or different excipients, and the like.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are meant to encompass any water-soluble poly(ethylene oxide).Typically, PEGs for use in accordance with the invention comprise thefollowing structure “—O(CH₂CH₂O)_(m)—” where (m) is 2 to 4000. As usedherein, PEG also includes “—CH₂CH₂—O(CH₂CH₂O)_(m)—CH₂CH₂—” and“—(CH₂CH₂O)_(m)—,” depending upon whether or not the terminal oxygenshave been displaced. When the PEG further comprises a spacer moiety (tobe described in greater detail below), the atoms comprising the spacermoiety, when covalently attached to a water-soluble polymer segment, donot result in the formation of an oxygen-oxygen bond (i.e., an “—O—O—”or peroxide linkage). Throughout the specification and claims, it shouldbe remembered that the term “PEG” includes structures having variousterminal or “end capping” groups and so forth. The term “PEG” also meansa polymer that contains a majority, that is to say, greater than 50%, of—CH₂CH₂O— monomeric subunits. With respect to specific forms, the PEGcan take any number of a variety of molecular weights, as well asstructures or geometries such as “branched,” “linear,” “forked,”“multifunctional,” and the like, to be described in greater detailbelow.

The terms “end-capped” or “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus, examples ofend-capping moieties include alkoxy (e.g., methoxy, ethoxy andbenzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. In addition, saturated, unsaturated, substituted and unsubstitutedforms of each of the foregoing are envisioned. Moreover, the end-cappinggroup can also be a silane. The end-capping group can alsoadvantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) of interest towhich the polymer is coupled can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, calorimetric (e.g.,dyes), metal ions, radioactive moieties, and the like. Suitabledetectors include photometers, films, spectrometers, and the like.

“Non-naturally occurring” with respect to a polymer or water-solublepolymer means a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer or water-soluble polymer may, however,contain one or more subunits or portions of a subunit that are naturallyoccurring, so long as the overall polymer structure is not found innature.

The term “water-soluble polymer” is any polymer that is soluble in waterat room temperature. Typically, a water-soluble polymer will transmit atleast about 75%, more preferably at least about 95% of light,transmitted by the same solution after filtering. On a weight basis, awater-soluble polymer will preferably be at least about 35% (by weight)soluble in water, more preferably at least about 50% (by weight) solublein water, still more preferably about 70% (by weight) soluble in water,and still more preferably about 85% (by weight) soluble in water. It isstill more preferred, however, that the water-soluble polymer is about95% (by weight) soluble in water and most preferred that thewater-soluble polymer is completely soluble in water.

Molecular weight in the context of a water-soluble polymer of theinvention, such as PEG, can be expressed as either a number averagemolecular weight or a weight average molecular weight. Unless otherwiseindicated, all references to molecular weight herein refer to the weightaverage molecular weight. Both molecular weight determinations, numberaverage and weight average, can be measured using gel permeationchromatography or other liquid chromatography techniques. Other methodsfor measuring molecular weight values can also be used, such as the useof end-group analysis or the measurement of colligative properties(e.g., freezing-point depression, boiling-point elevation, or osmoticpressure) to determine number average molecular weight or the use oflight scattering techniques, ultracentrifugation or viscometry todetermine weight average molecular weight. The polymers of the inventionare typically polydisperse (i.e., number average molecular weight andweight average molecular weight of the polymers are not equal),possessing low polydispersity values of preferably less than about 1.2,more preferably less than about 1.15, still more preferably less thanabout 1.10, yet still more preferably less than about 1.05, and mostpreferably less than about 1.03.

As used herein, the term “carboxylic acid” is a moiety having a

functional group [also represented as a “—COOH” or —C(O)OH], as well asmoieties that are derivatives of a carboxylic acid, such derivativesincluding, for example, protected carboxylic acids. Thus, unless thecontext clearly dictates otherwise, the term carboxylic acid includesnot only the acid form, but corresponding esters and protected forms aswell. With regard to protecting groups suited for a carboxylic acid andany other functional group described herein, reference is made to Greeneet al., “PROTECTIVE GROUPS IN ORGANIC SYNTHESIS” 3^(rd) Edition, JohnWiley and Sons, Inc., New York, 1999.

The terms “reactive” and “activated” when used in conjunction with aparticular functional group, refer to a reactive functional group thatreacts readily with an electrophile or a nucleophile on anothermolecule. This is in contrast to those groups that require strongcatalysts or highly impractical reaction conditions in order to react(i.e., a “nonreactive” or “inert” group).

The terms “protected,” “protecting group,” and “protective group” referto the presence of a moiety (i.e., the protecting group) that preventsor blocks reaction of a particular chemically reactive functional groupin a molecule under certain reaction conditions. The protecting groupwill vary depending upon the type of chemically reactive functionalgroup being protected as well as the reaction conditions to be employedand the presence of additional reactive or protecting groups in themolecule, if any. Protecting groups known in the art can be found inGreene et al., supra.

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof.

The terms “spacer” or “spacer moiety” are used herein to refer to anatom or a collection of atoms optionally appearing between one moietyand another. The spacer moieties may be hydrolytically stable or mayinclude one or more physiologically hydrolyzable or enzymaticallyreleasable linkages.

An “organic radical” as used herein includes, for example, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, aryl and substituted aryl.

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to20 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl when three or more carbon atoms are referenced and loweralkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, iso-butyl, and tert-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or Spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8carbon atoms.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenon-interfering substituents, such as, but not limited to: C₃-C₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like, for one or more hydrogen atoms.“Substituted aryl” is aryl having one or more non-interfering groups asa substituent. For substitutions on a phenyl ring, the substituents maybe in any orientation (i.e., ortho, meta, or para). “Substitutedammonium” is ammonium having one or more non-interfering groups (e.g.,an organic radical) as a substituent.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, etc.), more preferably C₁-C₇ alkyl.

As used herein, “alkenyl” refers to a branched or unbranched hydrocarbongroup of 2 to 15 atoms in length, containing at least one double bond.Exemplary alkenyl include (without limitation) ethenyl, n-propenyl,isopropenyl, n-butenyl, iso-butenyl, octenyl, decenyl, tetradecenyl, andthe like.

The term “alkynyl” as used herein refers to a branched or unbranchedhydrocarbon group of 2 to 15 atoms in length, containing at least onetriple bond. Exemplary alkynyl include (without limitation) ethynyl,n-butynyl, iso-pentynyl, octynyl, decynyl, and so forth.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl, or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl. Anaromatic-containing moiety (e.g., Ar¹, Ar², and so forth), means astructure containing aryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably N, O, or S, or a combination thereof. Heteroaryl rings mayalso be fused with one or more cyclic hydrocarbon, heterocyclic, aryl,or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom which is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or morenon-interfering groups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from non-interfering substituents.

“Electrophile” refers to an ion or atom or collection of atoms, that maybe ionic, having an electrophilic center, i.e., a center that iselectron seeking, capable of reacting with a nucleophile.

“Nucleophile” refers to an ion or atom or collection of atoms that maybe ionic having a nucleophilic center, i.e., a center that is seeking anelectrophilic center or with an electrophile.

A “physiologically cleavable” as well as a “hydrolyzable” bond is arelatively weak bond that reacts with water (i.e., is hydrolyzed) underphysiological conditions. The tendency of a bond to hydrolyze in waterwill depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Exemplary hydrolyzable bonds include, but are not limited to,carboxylate ester, phosphate ester, anhydride, acetal, ketal,acyloxyalkyl ether, imine, and ortho esters.

A “releasable linkage” includes, but is not limited to, aphysiologically cleavable bond, a hydrolyzable bond, and anenzymatically degradable linkage. Thus, a “releasable linkage” is alinkage that may undergo either hydrolysis or cleavage by some othermechanism (e.g., enzyme-catalyzed, acid-catalyzed, base-catalyzed, andso forth) under physiological conditions. For example, a “releaseablelinkage” can involve an elimination reaction that has a base abstractionof a proton, (e.g., an ionizable hydrogen atom, H_(α)), as the drivingforce. For purposes herein, a “releaseable linkage” is synonymous with a“degradable linkage.”

An “enzymatically releasable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, that is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include but are not limited to thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, and the like. Generally, a hydrolytically stable linkage is onethat exhibits a rate of hydrolysis of less than about 1-2% per day underphysiological conditions. Hydrolysis rates of representative chemicalbonds can be found in most standard chemistry textbooks. It must bepointed out that some linkages can be hydrolytically stable orhydrolyzable, depending upon (for example) adjacent and neighboringatoms and ambient conditions. One of ordinary skill in the art candetermine whether a given linkage or bond is hydrolytically stable orhydrolyzable in a given context by, for example, placing alinkage-containing molecule of interest under conditions of interest andtesting for evidence of hydrolysis (e.g., the presence and amount of twomolecules resulting from the cleavage of a single molecule). Otherapproaches known to those of ordinary skill in the art for determiningwhether a given linkage or bond is hydrolytically stable or hydrolyzablecan also be used.

The terms “active agent,” “biologically active agent” and“pharmacologically active agent” are used interchangeably herein and aredefined to include any agent, drug, compound, composition of matter ormixture that provides some pharmacologic, often beneficial, effect thatcan be demonstrated in vivo or in vitro. This includes food supplements,nutrients, nutriceuticals, drugs, proteins, vaccines, antibodies,vitamins, and other beneficial agents. As used herein, these termsfurther include any physiologically or pharmacologically activesubstance that produces a localized or systemic effect in a patient.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to an excipient that can be included in the compositionsof the invention and that causes no significant adverse toxicologicaleffects to the patient.

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a polymer-active agent conjugate—typicallypresent in a pharmaceutical preparation—that is needed to provide adesired level of active agent and/or conjugate in the bloodstream or ina target tissue. The exact amount will depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the pharmaceutical preparation, intended patientpopulation, patient considerations, and the like, and can readily bedetermined by one of ordinary skill in the art, based upon theinformation provided herein and available in the relevant literature.

“Multifunctional” in the context of a polymer means a polymer having 3or more functional groups contained therein, where the functional groupsmay be the same or different. Multifunctional polymers will typicallycontain from about 3-100 functional groups, or from 3-50 functionalgroups, or from 3-25 functional groups, or from 3-15 functional groups,or from 3 to 10 functional groups, or will contain 3, 4, 5, 6, 7, 8, 9or 10 functional groups within the polymer. A “difunctional” polymermeans a polymer having two functional groups contained therein, eitherthe same (i.e., homodifunctional) or different (i.e.,heterodifunctional).

“Branched,” in reference to the geometry or overall structure of apolymer, refers to polymer having 2 or more polymer “arms.” A branchedpolymer may possess 2 polymer arms, 3 polymer arms, 4 polymer arms, 6polymer arms, 8 polymer arms or more. One particular type of highlybranched polymer is a dendritic polymer or dendrimer, which, for thepurposes of the invention, is considered to possess a structure distinctfrom that of a branched polymer.

A “dendrimer” or dendritic polymer is a globular, size monodispersepolymer in which all bonds emerge radially from a central focal point orcore with a regular branching pattern and with repeat units that eachcontribute a branch point. Dendrimers exhibit certain dendritic stateproperties such as core encapsulation, making them unique from othertypes of polymers.

A basic or acidic reactant described herein includes neutral, charged,and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as provided herein, and includes both humans and animals.

As used herein, “drug release rate” means a rate (stated as a half-life)in which half of the total amount of polymer-active agent conjugates ina system will cleave into the active agent and a polymeric residue.

“Optional” and “optionally” mean that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

As used herein, the “halo” designator (e.g., fluoro, chloro, iodo,bromo, and so forth) is generally used when the halogen is attached to amolecule, while the suffix “ide” (e.g., fluoride, chloride, iodide,bromide, and so forth) is used when the halogen exists in itsindependent ionic form (e.g., such as when a leaving group leaves amolecule).

In the context of the present discussion, it should be recognized thatthe definition of a variable provided with respect to one structure orformula is applicable to the same variable repeated in a differentstructure, unless the context dictates otherwise.

As previously stated, the present invention comprises (among otherthings) conjugates having a releasable linkage.

Before describing exemplary conjugates of the invention, embodiments ofa water-soluble polymer and a functional group capable of reacting withan amino group of an active agent to form a releasable linkage, such asa carbamate linkage, will be discussed.

With respect to a given water-soluble polymer, each water-solublepolymer (e.g., POLY, POLY¹ and POLY²) can comprise any polymer so longas the polymer is water-soluble and non-peptidic. Although preferably apoly(ethylene glycol), a water-soluble polymer for use herein can be,for example, other water-soluble polymers such as other poly(alkyleneglycols) [also referred to as “poly(alkyleneoxides)”], such aspoly(propylene glycol) (“PPG”), copolymers of ethylene glycol andpropylene glycol and the like, poly(olefinic alcohol),poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), such as described in U.S. Pat. No.5,629,384. The water soluble polymer can be a homopolymer, copolymer,terpolymer, nonrandom block polymer, and random block polymer of any ofthe foregoing. In addition, a water-soluble polymer can be linear, butcan also be in other forms (e.g., branched, forked, and the like) aswill be described in further detail below. In the context of beingpresent within an overall structure, a water-soluble polymer has from 1to about 300 termini.

In instances where the polymeric reagent comprises two or morewater-soluble polymers, each water-soluble polymer in the overallstructure can be the same or different. It is preferred, however, thatall water-soluble polymers in the overall structure are of the sametype. For example, it is preferred that all water-soluble polymerswithin a given structure are poly(ethylene glycol) polymers.

Although the weight-average molecular weight of any individualwater-soluble polymer can vary, the weight average molecular weight ofany given water-soluble polymer will typically be in the followingrange: 100 Daltons to about 150,000 Daltons. Exemplary ranges, however,include weight-average molecular weights in the following ranges: in therange of from about 880 Daltons to about 5,000 Daltons; in the range ofgreater than 5,000 Daltons to about 100,000 Daltons; in the range offrom about 6,000 Daltons to about 90,000 Daltons; in the range of fromabout 10,000 Daltons to about 85,000 Daltons; in the range of greaterthan 10,000 Daltons to about 85,000 Daltons; in the range of from about20,000 Daltons to about 85,000 Daltons; in the range of from about53,000 Daltons to about 85,000 Daltons; in the range of from about25,000 Daltons to about 120,000 Daltons; in the range of from about29,000 Daltons to about 120,000 Daltons; in the range of from about35,000 Daltons to about 120,000 Daltons; in the range of about 880Daltons to about 60,000 Daltons; in the range of about 440 Daltons toabout 40,000 Daltons; in the range of about 440 Daltons to about 30,000Daltons; and in the range of from about 40,000 Daltons to about 120,000Daltons. For any given water-soluble polymer, PEGs having a molecularweight in one or more of these ranges are preferred.

Exemplary weight-average molecular weights for the water-soluble polymerinclude about 100 Daltons, about 200 Daltons, about 300 Daltons, about400 Daltons, about 440 Daltons, about 500 Daltons, about 600 Daltons,about 700 Daltons, about 750 Daltons, about 800 Daltons, about 900Daltons, about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons,about 2,200 Daltons, about 2,500 Daltons, about 3,000 Daltons, about4,000 Daltons, about 4,400 Daltons, about 4,500 Daltons, about 5,000Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons,about 7,500 Daltons, about 8,000 Daltons, about 9,000 Daltons, about10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000Daltons, about 14,000 Daltons, about 15,000 Daltons, about 16,000Daltons, about 17,000 Daltons, about 18,000 Daltons, about 19,000Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000Daltons, about 60,000 Daltons, about 65,000 Daltons, about 70,000Daltons, and about 75,000 Daltons. Branched versions of thewater-soluble polymer (e.g., a branched 40,000 Dalton water-solublepolymer comprised of two 20,000 Dalton polymers) having a total weightaverage molecular weight of any of the foregoing can also be used.

The polymeric reagent used to prepare the conjugate will comprise atleast one water-soluble polymer having a total size in the range suitedfor the desired rate of release of the conjugate formed therefrom. Forexample, a conjugate having a relatively long release rate can beprepared from a polymeric reagent having a size suited for (a) extendedcirculation prior to release of the active agent from the conjugate, and(b) moderately rapid in vivo clearance of the species liberated from theconjugate upon release from the conjugate. Likewise, when the conjugatehas a relatively fast release rate, then the polymeric reagent wouldtypically have a lower molecular weight.

When a PEG is used as the water-soluble polymer(s) in the polymericreagent, the PEG typically comprises a number of (OCH₂CH₂) monomers [or(CH₂CH₂O) monomers, depending on how the PEG is defined]. As usedthroughout the description, the number of repeating units is identifiedby the subscript “n” in “(OCH₂CH₂)_(n).” Thus, the value of (n)typically falls within one or more of the following ranges: from 2 toabout 3400, from about 4 to about 1500, from about 100 to about 2300,from about 100 to about 2270, from about 136 to about 2050, from about225 to about 1930, from about 450 to about 1930, from about 1200 toabout 1930, from about 568 to about 2727, from about 660 to about 2730,from about 795 to about 2730, from about 795 to about 2730, from about909 to about 2730, and from about 1,200 to about 1,900. For any givenpolymer in which the molecular weight is known, it is possible todetermine the number of repeating units (i.e., “n”) by dividing thetotal weight-average molecular weight of the polymer by the molecularweight of the repeating monomer.

Each water-soluble polymer is typically biocompatible andnon-immunogenic. With respect to biocompatibility, a substance isconsidered biocompatible if the beneficial effects associated with useof the substance alone or with another substance (e.g., an active agent)in connection with living tissues (e.g., administration to a patient)outweighs any deleterious effects as evaluated by a clinician, e.g., aphysician. With respect to non-immunogenicity, a substance is considerednon-immunogenic if use of the substance alone or with another substancein connection with living tissues does not produce an immune response(e.g., the formation of antibodies) or, if an immune response isproduced, that such a response is not deemed clinically significant orimportant as evaluated by a clinician. It is particularly preferred thatthe water-soluble polymers described herein as well as conjugates ofactive agents and the polymers are biocompatible and non-immunogenic.

In one form useful, free or nonbound PEG is a linear polymer terminatedat each end with hydroxyl groups:

HO—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—OH

wherein (m′) typically ranges from zero to about 4,000, preferably fromabout 20 to about 1,000.

The above polymer, alpha-,omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG- symbol can represent the following structural unit:

—CH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—

where (m′) is as defined as above.

Another type of free or nonbound PEG useful in the present invention ismethoxy-PEG-OH, or mPEG in brief, in which one terminus is therelatively inert methoxy group, while the other terminus is a hydroxylgroup. The structure of mPEG is given below.

CH₃OCH₂CH₂O—(CH₂CH₂O)_(m′)—CH₂CH₂—

where (m′) is as described above.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, can also be used as the PEG polymer. For example,PEG can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones (either the same or different),such as methoxy poly(ethylene glycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine.

In addition, the PEG can comprise a forked PEG. An example of a free ornonbound forked PEG is represented by the following formula:

wherein: X is a spacer moiety and each Z is an activated terminal grouplinked to CH by a chain of atoms of defined length. The chain of atomslinking the Z functional groups to the branching carbon atom serve as atethering group and may comprise, for example, alkyl chains, etherchains, ester chains, amide chains and combinations thereof. U.S. Pat.No. 6,362,254, discloses various forked PEG structures capable of use inthe present invention.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG rather than at the end of the PEG chain. The pendant reactive groupscan be attached to the PEG directly or through a spacer moiety, such asan alkylene group.

In addition to the above-described forms of PEG, each water-solublepolymer in the polymeric reagent can also be prepared with one or moreweak or releasable linkages in the polymer, including any of the abovedescribed polymers. For example, PEG can be prepared with ester linkagesin the polymer that are subject to hydrolysis. As shown below, thishydrolysis results in cleavage of the polymer into fragments of lowermolecular weight:

-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically releasable linkages, useful as a releasable linkagewithin a polymer backbone, include carbonate linkages; imine linkagesresulting, for example, from reaction of an amine and an aldehyde (see,e.g., Ouchi et al. (1997) Polymer Preprints 38(1):582-3); phosphateester linkages formed, for example, by reacting an alcohol with aphosphate group; hydrazone linkages which are typically formed byreaction of a hydrazide and an aldehyde; acetal linkages that aretypically formed by reaction between an aldehyde and an alcohol; orthoester linkages that are, for example, formed by reaction between aformate and an alcohol; amide linkages formed by an amine group, e.g.,at an end of a polymer such as PEG, and a carboxyl group of another PEGchain; urethane linkages formed from reaction of, e.g., a PEG with aterminal isocyanate group and a PEG alcohol; peptide linkages formed byan amine group, e.g., at an end of a polymer such as PEG, and a carboxylgroup of a peptide; and oligonucleotide linkages formed by, for example,a phosphoramidite group, e.g., at the end of a polymer, and a 5′hydroxyl group of an oligonucleotide.

It is understood by those of ordinary skill in the art that the termpoly(ethylene glycol) or PEG represents or includes all the above formsof PEG.

Those of ordinary skill in the art will recognize that the foregoingdiscussion concerning substantially water-soluble polymers is by nomeans exhaustive and is merely illustrative, and that all polymericmaterials having the qualities described above are contemplated. As usedherein, the term “water-soluble polymer” refers both to a molecule aswell as the residue of water-soluble polymer that has been attached toanother moiety. The following description of a water-soluble polymer areapplicable not only to the polymeric reagent, but to the correspondingconjugates formed using the described polymeric reagents.

The functional group of the polymeric reagents used to form theconjugates described herein is a functional group capable of reactingwith an amino group of an active agent to form a releasable linkage,such as a carbamate linkage. The invention is not limited with respectto the specific functional group so long as the functional group iscapable of reacting with an amino group of an active agent to form areleasable linkage, such as a carbamate linkage. Exemplary functionalgroups capable of reacting with an amino group of an active agentinclude those functional groups selected from the group consisting ofactive carbonates such as N-succinimidyl, 1-benzotriazolyl, imidazole,carbonate halides (such as carbonate chloride and carbonate bromide),phenolates (such as p-nitrophenolate) and so forth. Also, as a specialcase, if the active agent is available with the active amine groupconverted into an isocyanate or isothiocyanate group, then thefunctional group of the polymeric reagent can be hydroxyl as thereaction of these components provides a releasable carbamate linkage.

Exemplary polymeric reagents will now be discussed in further detail. Itmust be remembered that while stereochemistry is not specifically shownin any formulae or structures (whether for a polymeric reagent,conjugate, or any other formula or structure), the provided formulae andstructures contemplate both enantiomers, as well as compositionscomprising mixtures of each enantiomer in equal amounts (i.e., a racemicmixture) and unequal amounts.

An exemplary polymeric reagent has the following structure:

wherein:

POLY¹ is a first water-soluble polymer;

POLY² is a second water-soluble polymer;

X¹ is a first spacer moiety;

X² is a second spacer moiety;

H_(α) is an ionizable hydrogen atom;

R¹ is H or an organic radical;

R² is H or an organic radical;

(a) is either zero or one;

(b) is either zero or one;

R^(e1), when present, is a first electron altering group;

R^(e2), when present, is a second electron altering group; and

(FG) is a functional group capable of reacting with an amino group of anactive agent to form a releasable linkage, such as a carbamate linkage.

Exemplary polymeric reagents fall within the following formulae:

wherein, in each instance: (FG) is a functional group capable ofreacting with an amino group of an active agent to form a releasablelinkage, such as a carbamate linkage; R¹ is H or an organic radical; andR² is H or an organic radical.

Still another exemplary polymeric reagents have the structure:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and R^(e1) is a first electron altering group; andR^(e2) is a second electron altering group.

Still another exemplary polymeric reagents fall within the followingstructures

wherein, for each structure and in each instance, (n) is independentlyan integer from 4 to 1500.

The polymeric reagents can be prepared in any number of ways.Consequently, synthesis of the polymeric reagents is not limited to thespecific technique or approach used in their preparation.

In one method for preparing a polymeric reagent useful in preparing theconjugates described herein, the method comprises: (a) providing anaromatic-containing moiety bearing a first attachment site, a secondattachment site and an optional third attachment site; (b) reacting afunctional group reagent with the first attachment site to result in thefirst attachment site bearing a functional group capable of reactingwith an amino group of an active agent and result in a releasablelinkage, such as a carbamate; and (c) reacting a water-soluble polymerbearing a reactive group with the second attachment site and, whenpresent, the optional third attachment site to result in (i) the secondattachment site bearing a water-soluble polymer through a spacer moietyand (ii) the optional third attachment site, when present, bearing asecond water-soluble polymer through a spacer moiety. In some instances,(b) is performed before step (c) while in other instances, (c) isperformed before step (b).

Thus, in this method for preparing a polymeric reagent, a required stepis (a) providing an aromatic-containing moiety bearing a firstattachment site, a second attachment site and an optional thirdattachment site. In the context of a synthetic preparation, it isunderstood that “providing” a material means to obtain the material (by,for example, synthesizing it or obtaining it commercially). An exemplaryaromatic-containing moiety, for illustrative purposes, is9-hydroxymethyl-2,7-diaminofluorene, as shown below.

This aromatic-containing moiety, 9-hydroxymethyl-2,7-diaminofluorene, isan example of an aromatic-containing moiety having three attachmentsites: a hydroxyl group at the 9 position and amino groups at each ofthe 2 and 7 positions. The aromatic-containing moiety can be provided ina base or salt form. With respect to9-hydroxymethyl-2,7-diaminofluorene, it is possible to use thedihydrochloride form. Other aromatic-containing moieties can be providedvia synthetic preparation and/or purchase from a commercial supplier.

Having provided the aromatic-containing moiety, another step in themethod broadly includes the step of reacting a water-soluble polymerbearing a reactive group with the attachment site(s) on thearomatic-containing moiety. Here, any art-known approach for attaching awater-soluble polymer to one or more attachment sites on thearomatic-containing moiety can be used and the method is not limited tothe specific approach. For example, an amine-reactive PEG (such as anN-succinimidyl ester-terminated mPEG, formed, for example, from thereaction of N-hydroxysuccinimide andCH₃O—CH₂CH₂—(OCH₂CH₂)—OCH₂CH₂—OCH₂COOH with dicyclohexyl carbodiimide(DCC) or diisopropyl carbodiimide (DIC) as a condensing agent andoptionally in the presence of a base) can be reacted with an aminebearing aromatic-containing moiety such as9-hydroxymethyl-2,7-diaminofluorene.

In some instances, reaction of the water-soluble polymer bearing areactive group with the aromatic-containing moiety will result in allpossible attachment sites having water-soluble polymer attached thereto.In such circumstances it is necessary to remove at least onewater-soluble polymer so that an attachment site is made available forreaction with a functional group reagent. Thus, for example, reaction ofthe N-succinimidyl ester-terminated mPEG discussed in the previousparagraph with 9-hydroxymethyl-2,7-diaminofluorene results in a mixturecomprising (a) a species bearing two water-soluble polymers, one at eachof the two amine sites, and (b) a species bearing three water-solublepolymers, one at each of the two amine sites, and one at the hydroxylsite. Here, it is possible to remove and collect higher molecular weightspecies by using size-exclusion chromatography. In addition it ispossible to treat the mixture to high pH [treating, for example, themixture to lithium hydroxide (LiOH), sodium hydroxide (NaOH), potassiumhydroxide (KOH)], followed by ion-exchange chromatography (IEC). Ineither case, the result is a composition containing mostly9-hydroxymethyl-2,7-diaminofluorene bearing two water-soluble polymers,one at each of the two amine sites. A third hydroxyl site is therebyavailable for reaction with a functional group reagent.

The final step is reacting a reactive site of the aromatic-containingmoiety with a functional group reagent. A preferred approach is to reactthe hydroxyl-containing 9-hydroxymethyl-2,7-diaminofluorene bearing twowater-soluble polymers, one at each of the two amine sites withtriphosgene followed by treatment with N-hydroxysuccinimide. In thisway, a functional group capable of reacting with an amino group of anactive agent to form a releasable linkage, such as a carbamate linkage(in this case, an “activated carbonate”) is formed on thehydroxyl-containing reactive site.

No matter which approach is used, the steps of the synthetic method takeplace in an appropriate solvent. One of ordinary skill in the art candetermine whether any specific solvent is appropriate for any givenreaction. Typically, however, the solvent is preferably a nonpolarsolvent or a polar aprotic solvent. Nonlimiting examples of nonpolarsolvents include benzene, xylene, dioxane, tetrahydrofuran (THF),t-butyl alcohol and toluene. Particularly preferred nonpolar solventsinclude toluene, xylene, dioxane, tetrahydrofuran, and t-butyl alcohol.Exemplary polar aprotic solvents include, but are not limited to, DMSO(dimethyl sulfoxide), HMPA (hexamethylphosphoramide), DMF(dimethylformamide), DMA (dimethylacetamide), NMP(N-methylpyrrolidinone).

Once prepared, the polymeric reagents can be isolated. Known methods canbe used to isolate the polymeric reagent, but it is particularlypreferred to use chromatography, e.g., size exclusion chromatography.Alternately or in addition, the method includes the step of purifyingthe polymeric reagent once it is formed. Again, standard art-knownpurification methods can be used to purify the polymeric reagent.

The polymeric reagents are sensitive to moisture and oxygen and areideally stored under an inert atmosphere, such as under argon or undernitrogen, and at low temperature. In this way, potentially degradativeprocesses associated with, for example, atmospheric oxygen, are reducedor avoided entirely. In some cases, to avoid oxidative degradation,antioxidants, such as butylated hydroxyl toluene (BHT), can be added tothe polymeric reagent prior to storage. In addition, it is preferred tominimize the amount of moisture associated with the storage conditionsto reduce potentially damaging reactions associated with water, e.g.,hydrolysis of the active ester. Moreover, it is preferred to keep thestorage conditions dark in order to prevent certain degradativeprocesses that involve light. Thus, preferred storage conditions includeone or more of the following: storage under dry argon or another dryinert gas; storage at temperatures below about −15° C.; storage in theabsence of light; and storage with a suitable amount (e.g., about 50 toabout 500 parts per million) of an antioxidant such as BHT.

The above-described polymeric reagents are useful for conjugation tobiologically active agents. For example, an amino group (e.g., primaryamine) on an active agent will react with the functional group capableof reacting with an amino group of an active agent to form a releasablelinkage, such as a carbamate linkage.

Exemplary conjugates include those of the following formulae:

wherein:

POLY¹ is a first water-soluble polymer;

POLY² is a second water-soluble polymer;

X¹ is a first spacer moiety;

X² is a second spacer moiety;

H_(α) is an ionizable hydrogen atom;

R¹ is H or an organic radical;

R² is H or an organic radical;

(a) is either zero or one;

(b) is either zero or one;

R^(e1), when present, is a first electron altering group;

R^(e2), when present, is a second electron altering group;

Y¹ is O or S;

Y² is O or S; and

(vWF/F8) is a residue of a amine-containing biologically active agentselected from the group consisting of a von Willebrand Factor moiety anda Factor VIII moiety.

Exemplary conjugates have the following structure:

wherein, for each structure and in each instance, (n) is independentlyan integer from 4 to 1500, and (vWF/F8) is a residue of a biologicallyactive agent selected from the group consisting of a von WillebrandFactor moiety and a Factor VIII moiety.

The biologically active agent to which a polymeric reagent as describedherein can be conjugated, is an amine-containing biologically activeagent. Typically, the biologically active agent will be a macromolecule,such as a polypeptide, having a molecular weight greater than about3,500 Daltons. Pharmacologically active polypeptides represent apreferred type of biologically active agent. It should be understoodthat for purposes of the present discussion, the term “polypeptide” willbe generic for oligopeptides and proteins. With regard to polypeptides,the amine to which the polymeric reagent couples to can be on theN-terminus or an amine-containing side chain of an amino acid (such aslysine) within the polypeptide.

The invention also provides for a method of preparing a conjugatecomprising the step of contacting a polymeric reagent with abiologically active agent under conditions suitable to form a covalentattachment between the polymer and the biologically active agent.Typically, the polymer is added to the active agent or surface at anequimolar amount (with respect to the desired number of groups suitablefor reaction with the reactive group) or at a molar excess. For example,the polymeric reagent can be added to the target active agent at a molarratio of about 1:1 (polymeric reagent:active agent), 1.5:1, 2:1, 3:1,4:1, 5:1, 6:1, 8:1, or 10:1. The conjugation reaction is allowed toproceed until substantially no further conjugation occurs, which cangenerally be determined by monitoring the progress of the reaction overtime. Progress of the reaction can be monitored by withdrawing aliquotsfrom the reaction mixture at various time points and analyzing thereaction mixture by SDS-PAGE or MALDI-TOF mass spectrometry or any othersuitable analytical method. Once a plateau is reached with respect tothe amount of conjugate formed or the amount of unconjugated polymerremaining, the reaction is assumed to be complete. Typically, theconjugation reaction takes anywhere from minutes to several hours (e.g.,from 5 minutes to 24 hours or more). The resulting product mixture ispreferably, but not necessarily, purified to separate out excessreagents, unconjugated reactants (e.g., active agent) undesiredmulti-conjugated species, and free or unreacted polymer. The resultingconjugates can then be further characterized using analytical methodssuch as MALDI, capillary electrophoresis, gel electrophoresis, and/orchromatography.

It is possible to characterize the degree of attachment (that is, thenumber—often expressed in terms of an average number in the context of acomposition of conjugates—of polymeric reagents that became attached tothe protein) of a conjugate. To determine the average number ofwater-soluble polymer molecules on protein conjugates, analyticaltechniques such as SDS-PAGE, SEC, IEC, MALDI-TOF, and so forth can beused. Spectrophotometric detection of the residual primary amine on aprotein using TNBSA following water-soluble polymer attachment has ledto the qualitative and quantitative estimation of degree of attachment.With respect to von Willebrand Factor-water-soluble polymer conjugates,the degree of attachment was qualitatively described as low, medium, andhigh based on SDS-PAGE. When analyzing the degree of attachment ofreleasable von Willebrand Factor-water-soluble polymer conjugates bySDS-PAGE under reducing conditions, release of the water-soluble polymerduring sample treatment and electrophoresis can lead to an overallunderestimate of the true degree of PEGylation.

One approach for the determination of the degree of PEG-based polymersattachment is a modification of the work of Nag and Barker. See Nag etal. (1996) Anal. Biochem. 237:224-231 and Barker et al. (2001) Anal.Biochem. 290:382-385. The method is based on principle of partitioningof a chromophore, ammonium ferrothiocyanate, from the aqueous phase intothe chloroform phase in the presence of PEG (as the chromophore itselfcan not be extracted into the chloroform phase without the PEGpresence). Digestion of von Willebrand Factor by pronase frees the PEGfrom the conjugate, and the quantity of the PEG is determinedsubsequently by the ammonium ferrothiocyanate method. Using thisprinciple, a reverse phase HPLC method that circumvents the need forchloroform extraction was developed to determine the degree ofattachment for the conjugates described herein.

With respect to polymer-active agent conjugates, the conjugates can bepurified to obtain/isolate different conjugated species. Alternatively,and more preferably for lower molecular weight (e.g., less than about 20kiloDaltons, more preferably less than about 10 kiloDaltons) polymers,the product mixture can be purified to obtain the distribution ofwater-soluble polymer segments per active agent. For example, theproduct mixture can be purified to obtain an average of anywhere fromone to five PEGs per active agent (e.g., polypeptide). The strategy forpurification of the final conjugate reaction mixture will depend upon anumber of factors, including, for example, the molecular weight of thepolymer employed, the particular active agent, the desired dosingregimen, and the residual activity and in vivo properties of theindividual conjugate(s).

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography. That is to say, gelfiltration chromatography is used to fractionate differently numberedpolymer-to-active agent ratios (e.g., 1-mer, 2-mer, 3-mer, and so forth,wherein “1-mer” indicates 1 polymer to active agent, “2-mer” indicatestwo polymers to active agent, and so on) on the basis of their differingmolecular weights (where the difference corresponds essentially to theaverage molecular weight of the water-soluble polymer segments). Forexample, in an exemplary reaction where a 100 kDa protein is randomlyconjugated to a polymeric reagent having a molecular weight of about 20kDa, the resulting reaction mixture will likely contain unmodifiedprotein (MW 100 kDa), mono-PEGylated protein (MW 120 kDa), di-PEGylatedprotein (MW 140 kDa), and so forth. While this approach can be used toseparate PEG and other polymer conjugates having different molecularweights, this approach is generally ineffective for separatingpositional isomers having different polymer attachment sites within theprotein. For example, gel filtration chromatography can be used toseparate from each other mixtures of PEG 1-mers, 2-mers, 3-mers, and soforth, although each of the recovered PEG-mer compositions may containPEGs attached to different reactive amino groups (e.g., lysine residues)within the active agent.

Gel filtration columns suitable for carrying out this type of separationinclude Superdex™ and Sephadex™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) optical density (OD) at280 nm for protein content, (ii) bovine serum albumin (BSA) proteinanalysis, (iii) iodine testing for PEG content [Sims et al.(1980) Anal.Biochem, 107:60-63], and (iv) sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS PAGE), followed by staining with barium iodide.

Separation of positional isomers is carried out by reverse phasechromatography using a reverse phase-high performance liquidchromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac) orby ion exchange chromatography using an ion exchange column, e.g., aSepharose™ ion exchange column available from Amersham Biosciences.Either approach can be used to separate polymer-active agent isomershaving the same molecular weight (positional isomers).

An amine-containing biologically active agent for use in coupling to apolymer as presented herein may be a von Willebrand Factor moiety or aFactor VIII moiety.

With respect to a von Willebrand Factor moiety (“vWF”), the vonWillebrand Factor moiety useful for the present invention includes anyprotein that has the same activity (although not necessarily the samedegree of activity) as native, human von Willebrand Factor and includesall forms of native, human von Willebrand Factor, including themonomeric and multimeric forms. Useful forms include homomultimers of atleast two von Willebrand Factors. The von Willebrand Factor moiety maybe either a biologically active derivative, or when to be used solely asa stabilizer for Factor VIII, the von Willebrand Factor moiety may be ofa form that is not biologically active. It should also be understoodthat the present invention encompasses different forms of von WillebrandFactor moieties to be used in combination. For example, a compositionuseful for the present invention may include different multimers,different derivatives and both biologically active derivatives andderivatives not biologically active.

The biologically activity of a von Willebrand Factor moiety can bemeasured in two different in vitro assays (Turecek et al., (2002) Semin.Thromb. Hemost. 28:149-160). The ristocetin cofactor assay is based onthe agglutination of fresh formalin-fixed platelets induced by theantibiotic ristocetin in the presence of a protein having von WillebrandFactor activity. The degree of platelet agglutination depends on theprotein concentration and can be measured by the turbidimetic method,e.g., by use of an aggregometer (Weiss et al. (1973) J. Clin. Invest.52:2708-2716; Macfarlane et al. (1975) Thromb. Diath. Haemorrh.34:306-308). The second method is the collagen binding assay, which isbased on ELISA technology (Brown et al. (1986) Thromb. Res. 43:303-311;Favaloro (2000) Thromb. Haemost. 83:127-135). A microtiter plate iscoated with type I or III collagen. The proposed von Willebrand Factormoiety is bound to the collagen surface and subsequently detected withan enzyme-labeled polyclonal antibody. The last step is the substratereaction, which can be photometrically monitored with an ELISA reader.Such methods are useful for determining the von Willebrand Factoractivity of both the moiety itself (and therefore can be used as a “vonWillebrand Factor moiety”) as well as the corresponding polymer-moietyconjugate.

Von Willebrand Factor- and Factor VIII-water-soluble polymer conjugatesare iologically active and exhibit increased in vivo half-lives ascompared to their corresponding non-conjugated versions. The increase inthe in vivo half-life can be assessed by measuring the pharmacokineticsof the conjugate, von Willebrand Factor, and Factor VIII in Factor VIIIdeficient mice as described in Examples 5 and 6 below. Briefly, FactorVIII deficient mice are treated with a bolus injection of von WillebrandFactor, or a von Willebrand Factor- or Factor VIII-water-soluble polymerconjugate, premixed with Factor VIII via the tail vein, and vonWillebrand Factor antigen levels are measured in plasma samples atvarious time points. In addition, Factor VIII deficient mice can betreated with a bolus injection of Factor VIII, or a von WillebrandFactor- or Factor VIII-water-soluble polymer conjugate, and Factor VIIIantigen levels are measured in plasma samples at various time points.Von Willebrand Factor antigen and Factor VIII antigen can be measuredvia ELISA assay.

The von Willebrand Factor moiety includes plasma-derived von WillebrandFactor and recombinant von Willebrand Factor. The von Willebrand Factormoiety may be produced by any method known in the art. One specificexample is disclosed in WO 86/06096.

With respect to a Factor VIII moiety, the Factor VIII moiety useful forthe present invention includes any protein that has the same activity(although not necessarily the same degree of activity) as native, humanFactor VIII. Included as a possible Factor VIII moiety is native, humanFactor VIII, which is a 2,351 amino acid, single chain glycoprotein thatis structurally organized as A1-A2-B-A3-C1-C2. When the expressedpolypeptide is translocated into the lumen of the endoplasmic reticulum,however, a 19-amino acid signal sequence is cleaved, resulting in asecond sequence. This second sequence, herein provided lacks the leading19 amino acids. It will be appreciated that a Factor VIII moiety is notlimited to merely “active” forms of Factor VIII (e.g., Factor VIIIa) andthat the term “Factor VIII moiety” encompasses “precursor” forms as wellas other substances that having a similar procoagulant effect.

For any given moiety, it is possible to determine whether that moietyhas Factor VIII activity. For example, several animal lines have beenintentionally bred with the genetic mutation for hemophilia such that ananimal produced from such a line has very low and insufficient levels ofFactor VIII. Such lines are available from a variety of sources such as,without limitation, the Division of Laboratories and Research, New YorkDepartment of Public Health, Albany, N.Y. and the Department ofPathology, University of North Carolina, Chapel Hill, N.C. Both of thesesources, for example, provide canines suffering from canine hemophiliaA. In order to test the Factor VIII activity of any given moiety inquestion, the moiety is injected into the diseased animal, a small cutmade and bleeding time compared to a untreated diseased animal as acontrol. Another method useful for determining Factor VIII activity isto determine cofactor and procoagulant activity. See, for example,Mertens et al. (1993) Brit. J Haematol. 85:133-42. Other methods knownto those of ordinary skill in the art can also be used to determinewhether a given moiety has Factor VIII activity. Such methods are usefulfor determining the Factor VIII activity of both the moiety itself (andtherefore can be used as a “Factor VIII moiety”) as well as thecorresponding polymer-moiety conjugate.

Nonlimiting examples of Factor VIII moieties include the following:Factor VIII; Factor VIIIa; Factor VIII:C; Factor VIII:vWF; B-domaindeleted Factor VIII (and other truncated versions of Factor VIII);hybrid proteins, such as those described in U.S. Pat. No. 6,158,888;glycosylated proteins having Factor VIII activity, such as thosedescribed in U.S. Patent Application Publication No. US2003/0077752; andpeptide mimetics having Factor VIII activity. Preferred truncated FactorVIII versions (encompassed by the term “B-domain deleted Factor VIII)corresponds to a protein having the amino acid sequence of human FactorVIII having a deletion corresponding to at least 581 amino acids withinthe region between Arg⁷⁵⁹ and Ser¹⁷⁰⁹, more preferably wherein thedeletion corresponds to one of the region between Pro¹⁰⁰⁰ and Asp¹⁵⁸²,the region between Thr⁷⁷⁸ and Pro¹⁶⁵⁹, and the region between Thr⁷⁷⁸ andGlu¹⁶⁹⁴.

With respect to both the von Willebrand Factor and Factor VIII moieties,biologically active fragments, deletion variants, substitution variantsor addition variants of any of the foregoing that maintain at least somedegree of the desired von Willebrand or Factor VIII activity can also beused.

The active agent can advantageously be modified to include one or moreamino acid residues such as, for example, lysine, cysteine and/orarginine, in order to provide facile attachment of the polymer to anatom within the side chain of the amino acid. Techniques for addingamino acid residues are well known to those of ordinary skill in theart. Reference is made to J. March, Advanced Organic Chemistry:Reactions Mechanisms and Structure, 4th Ed. (New York:Wiley-Interscience, 1992).

The active agent can be obtained from blood-derived sources. Forexample, Factor VIII can be fractionated from human plasma usingprecipitation and centrifugation techniques known to those of ordinaryskill in the art. See, for example, Wickerhauser (1976) Transfusion16(4):345-350 and Slichter et al. (1976) Transfusion 16(6):616-626.Factor VIII can also be isolated from human granulocytes. See Szmitkoskiet al. (1977) Haematologia (Budap.) 11(1-2):177-187.

In addition, the active agent can also be obtained from recombinantmethods. Briefly, recombinant methods involve constructing the nucleicacid encoding the desired polypeptide or fragment, cloning the nucleicacid into an expression vector, transforming a host cell (e.g.,bacteria, yeast, or mammalian cell such as Chinese hamster ovary cell orbaby hamster kidney cell), and expressing the nucleic acid to producethe desired polypeptide or fragment. Methods for producing andexpressing recombinant polypeptides in vitro and in prokaryotic andeukaryotic host cells are known to those of ordinary skill in the art.See, for example, U.S. Pat. No. 4,868,122.

The above exemplary biologically active agents are meant to encompass,where applicable, analogues, agonists, antagonists, inhibitors, isomers,and pharmaceutically acceptable salt forms thereof. In reference topeptides and proteins, the invention is intended to encompass synthetic,recombinant, native, glycosylated, and non-glycosylated forms, as wellas biologically active fragments thereof. In addition, the term “activeagent” is intended to encompass the active agent prior to conjugation aswell as the active agent “residue” following conjugation.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Acids or bases may be present as an excipient in the preparation.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

The pharmaceutical preparations encompass all types of formulations andin particular those that are suited for injection, e.g., powders thatcan be reconstituted as well as suspensions and solutions. The amount ofthe conjugate (i.e., the conjugate formed between the active agent andthe polymer described herein) in the composition will vary depending ona number of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container (e.g., avial). In addition, the pharmaceutical preparation can be housed in asyringe. A therapeutically effective dose can be determinedexperimentally by repeated administration of increasing amounts of theconjugate in order to determine which amount produces a clinicallydesired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about5%-98% by weight, more preferably from about 15-95% by weight of theexcipient, with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”,19^(th) ed., Williams & Williams, (1995), the “Physician's DeskReference”, 52^(nd) ed., Medical Economics, Montvale, N.J. (1998), andKibbe, A. H., Handbook of Pharmaceutical Excipients, 3^(rd) Edition,American Pharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical preparations of the present invention are typically,although not necessarily, administered via injection and are thereforegenerally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, subcutaneous,intra-arterial, and so forth.

As previously described, the conjugates can be administered parenterallyby intravenous injection, or less preferably by intramuscular or bysubcutaneous injection. Suitable formulation types for parenteraladministration include ready-for-injection solutions, dry powders forcombination with a solvent prior to use, suspensions ready forinjection, dry insoluble compositions for combination with a vehicleprior to use, and emulsions and liquid concentrates for dilution priorto administration, among others.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering, generally via injection, a therapeutically effectiveamount of the conjugate (preferably provided as part of a pharmaceuticalpreparation). The method of administering may be used to treat anycondition that can be remedied or prevented by administration of theparticular conjugate. Those of ordinary skill in the art appreciatewhich conditions a specific conjugate can effectively treat. The actualdose to be administered will vary depend upon the age, weight, andgeneral condition of the subject as well as the severity of thecondition being treated, the judgment of the health care professional,and conjugate being administered. Therapeutically effective amounts areknown to those skilled in the art and/or are described in the pertinentreference texts and literature. Generally, a therapeutically effectiveamount will range from about 0.001 mg to 100 mg, preferably in dosesfrom 0.01 mg/day to 75 mg/day, and more preferably in doses from 0.10mg/day to 50 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the experimental that follow areintended to illustrate and not limit the scope of the invention. Otheraspects, advantages and modifications within the scope of the inventionwill be apparent to those skilled in the art to which the inventionpertains.

All articles, books, patents, patent publications and other publicationsreferenced herein are hereby incorporated by reference in theirentireties.

Experimental

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis and the like, which areunderstood by one of ordinary skill in the art and are explained in theliterature. In the following examples, efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperatures, andso forth), but some experimental error and deviation should be accountedfor. Unless otherwise indicated, temperature is in degrees Celsius andpressure is at or near atmospheric pressure at sea level. All reagentswere obtained commercially unless otherwise indicated. All generated NMRwas obtained from a 300 or 400 MHz NMR spectrometer manufactured byBruker (Billerica, Mass.). All processing is carried out in glass orglass-lined vessels and contact with metal-containing vessels orequipment is avoided.

The following abbreviations will be used.

FVIII; rFVIII factor VIII; recombinant FVIII

HPLC high pressure liquid chromatography

hydr hydrolyzable

PEG-rVWF PEGylated rVWF

PEGrFVIII PEGylated rFVIII

rVWF recombinant von Willebrand factor

rFVIII recombinant FVIII

SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis

The rVWF product used for PEGylation was a purified rVWF preparationderived from a Chinese hamster ovary (CHO) cell line and was purifiedusing conventional purification techniques.

Polymeric reagents were made in accordance with the basic approachesdescribed in U.S. Patent Application Publication No. 2006/0293499 andhad the following structures:

EXAMPLE 1A Preparation of vWF Coniugate (20,000 Da Total Polymer WeightAverage Molecular Weight) (“Lys 20K br Long”)

-   -   (wherein VWF is a residue of von Willebrand Factor)

An appropriate quantity of VWF protein solution was thawed (using warmwater of ±30° C.) so as to result in a protein solution having 60 mg ofprotein content. The protein solution was poured into a new sterilized400 mL disposable polypropylene beaker. If necessary, the temperature ofthe protein solution was adjusted to 22° C. (±1° C.). If necessary, theprotein solution was diluted with a solution [20 mM HEPES (pH 7.4), 150mM NaCl, 0.5% w/v sucrose] or concentrated to ensure a concentration of0.45 mg/mL±0.05 mg/mL. A sample of 0.2 mL was retained and stored at 4°C. for later concentration verification. The protein solution beaker wasplaced under an overhead stirrer, wherein the impeller was lowered intothe protein solution approximately ¾ down (i.e., ¼ from the bottom) andthe impeller set to stirring at 60rpm (±2 rpm). In order to preventcontamination as much as possible, the beaker was covered.

A seventy-five molar excess (relative to von Willebrand Factor monomermass of 278 kDa) of polymeric reagent B having a total polymer weightaverage molecular weight (i.e., the sum of the weight average molecularweight of each polymer “arm”) of about 20,000 Da was weighed and placedinto a 50 mL polypropylene Falcon tube and dissolved by adding 2 mM HClin an amount sufficient to provide a 5% w/v PEG or 50 mg/mL PEGsolution). Optionally, the PEG solution can be centrifuged (using aBeckman bench top centrifuge equipped with holders for 50 mL Falcontubes, at 1000 rpm) which will result in a clear solution collected atthe bottom of the tube. As soon as the PEG solution was formed, it waspumped via a syringe pump into the protein solution at a rate of 1.5mL/min (90 mL/h). The tube transporting the PEG solution was placed inthe beaker such that the PEG solution was fed into the protein solutionat the level of the impeller. Hereafter, the protein solution combinedwith the PEG solution is referred to as the “PEGylation reactionmixture”. Stirring of the PEGylation reaction mixture was continued forfive hours, with temperature (22° C.±1° C.) and pH monitored atintervals as required.

Following five hours of stirring (the pH of the PEGylation reactionsolution should be 7.3±0.1), 14.5 mL of a 0.1 M glycine solution wasadded (at 1.5 mL/min in the same way the PEG solution was added to thePEGylation reaction solution) to thereby form a glycine-containingPEGylation reaction mixture. The final concentration of glycine in theglycine-containing PEGylation reaction mixture should be 10 mM (±1 mM).The glycine-containing PEGylation reaction mixture was stirred at 60 rpmfor another two hours.

Following two hours of stirring, a 0.2 mL sample was removed and storedat 4° C. for protein determination.

To purify the conjugate within the glycine-containing PEGylationreaction mixture, the glycine-containing PEGylation reaction mixture wasdiluted with 3 glycine-containing PEGylation reaction volumes ofsolution A [20 mM sodium citrate (pH 6. 1), 0.5% w/v sucrose] to reducethe NaCl concentration below 100 mM and to dilute the unbound freepolymeric reagent B. Following dilution, the glycine-containingPEGylation reaction solution was mixed with gentle rotation (swirling)or mixing with an overhead stirrer. The conjugate was purified by cationexchange chromatography on an ÄKTA Basic System. A Millipore Vantage 44mm ID column packed with GE-Healthcare SP-HP media. The packed bedheight was 100-105 mm resulting in a column volume of 150-160 mL,thereby resulting in a column loading of ≦0.4 mg/mL. The flow rate inthe column was set to 15 mL/min (linear flow rate of 1 cm/min). Themobile phase used for the purification included solution A [20 mM sodiumcitrate (pH 6.1), 0.5% w/v sucrose] and solution B [20 mM sodium citrate(pH 6.1), 0.5% w/v sucrose, 1.0 M NaCl], or a mixture of both, whereinthe mobile phase was run using a gradient. The following gradient wasused: Step 1: 0% of the starting mobile phase contained solution B; Step2: for the first retention volume equaling 0.7 of the column volume, themobile phase contained 0 to 70% of solution B; Step 3: for the nextretention volume equaling 2.5 of the column volume, 70% of the mobilephase contained solution B. The UV absorbance of the eluent wasmonitored at 280 nm. The unbound free polymeric reagent B eluted duringStep 1. The conjugate, which eluted during Steps 2 and 3, was collectedas soon as the absorbance began to rise from the baseline and wasstopped when the peak diminished back to 7% of maximum peak height. Atypical chromatogram prepared in accordance with this procedure isprovided as FIG. 1.

EXAMPLE 1B Preparation of vWF Conjugate (40,000 Da Total Polymer WeightAverage Molecular Weight) (“Lys 40K br Long”)

The basic procedure of Example 1A was repeated except that polymericreagent B having a total polymer weight average molecular weight ofabout 40,000 Da was used instead of about 20,000 Da.

EXAMPLE 1C Preparation of vWF Conjugate (60,000 Da Total Polymer WeightAverage Molecular Weight) (“Lys 60K br Long”)

The basic procedure of Example 1A was repeated except that polymericreagent B having a total polymer weight average molecular weight ofabout 60,000 Da was used instead of about 20,000 Da.

EXAMPLE 2A Preparation of FVIII Coniugate (20,000 Da Total PolymerWeight Average Molecular Weight) (“Lys 20K br Long”)

-   -   (wherein FVIII is a residue of Factor VIII)

FVIII protein solution (3.23 mg/mL protein concentration) was quicklythawed (using a warm water bath at room temperature for five minutes)and, using a 1000 μL pipettor, approximately 3.1 mL of the warmed FVIIIprotein solution was placed in a 50 mL conical tube.

A 42.8 molar ratio (relative to Factor VIII) of polymeric reagent B (38mg) having a total polymer weight average molecular weight (i.e., thesum of the weight average molecular weight of each polymer “arm”) ofabout 20,000 Da was placed into a 2 mL microcentrifuge tube. The weighedpolymeric reagent B was suspended in 500 μL of 2 mM HCl. Polymericreagent B was solubilized by alternating and centrifuging themicrocentrifuge tube over a twenty second period.

Using a pipettor, the solution of polymeric reagent B so formed wasadded to the warmed FVIII protein solution dropwise over 10-20 seconds.The resulting mixture was maintained at room temperature (approximately22° C.) for one hour. At the end of one hour, 36 μL of a 0.1 M glycinesolution was added to thereby form a glycine-containing PEGylationreaction mixture. A 100 μL sample was placed in a 500 μL microcentrifugetube and then placed in a −80° C. freezer.

To remove salt within the glycine-containing PEGylation reactionmixture, a 5 mL HiTrap DeSalt column was pre-equilibrated with 20 mMhistidine, 10 mM CaCl₂, 0.1% Tween 80, pH 6.5]. Once equilibrated, theentire volume of the glycine-containing PEGylation reaction mixture wasloaded onto the column and fractions were collected and pooled.Protein-containing fractions were collected, placed in a container andimmediately placed in a standard ice bath.

To purify the conjugate within the glycine-containing PEGylationreaction mixture, the glycine-containing PEGylation reaction mixture wasdiluted 1:10 solution A [20 mM histidine, 10 mM CaCl₂, 0.1% Tween 80, pH6.5]. The conjugate was purified by cation exchange chromatography on anÄKTA Basic System. The column used was a 5 mL HiTrap Q HP column (systemand column washed with 0.1 M NaOH and complete removal of NaOH wasverified by testing for neutral or near neutral pH following washingwith Milli-Q water or purification buffer). The column was washed with10 mL of solution A at 2.0 mL/min and the flow through was collected in5 mL fractions. The mobile phase used for the purification includedsolution A, solution B [20 mM histidine, 10 mM CaCl₂, 0.1% Tween 80, pH6.5, 1 M NaCl], or a mixture of both, wherein the mobile phase was runusing a gradient. A column wash of 2 column volumes (10 mL) of solutionA was run. The following gradient was used: 0% of the starting mobilephase contained solution B; a step to 50% of solution B in the mobilewas used and held for 15 mL (the peak was collected in approximately 2mL fractions and were stored on ice); a step to 100% of solution B inthe mobile phase was used and held for 5 mL; and finally, a step back to0% of solution B in the mobile phase was used and held for 15 mL. Atypical separation profile is provided as FIG. 2A.

Protein determination was carried out by thawing 1×0.2 mL aliquot ofpurified conjugated sample and 100 μL/mL of Factor VIII. A standardcurve with points at 0.2, 0.5, 0.75 and 1.5 mg/mL of Factor VIII wasprepared. For each run (sample, standard, or purification buffer), 30 μLof the appropriate substance was placed in a clean 5 mL tube and 1.5 mLof Pierce Protein Assay Reagent (Pierce Biotechnology, Inc., RockfordIll.) was added to the tube and was followed by mixing of the contentsof the tube. After incubation for ten minutes at room temperature (22°C.), the contents of each tube were read using a spectrophotometer at595 nm.

Analysis via ion-exchange chromatography was carried out by placing a4.6×50 Mini Q column (GE Healthcare Bio-Sciences Corp, Piscataway N.J.)on an Agilent 1100 chromatography system (Agilent Technologies, Inc.,Santa Clara Calif.), wherein buffers were the same as those used forpurification and the maximum flow rate used was 0.5 mL/minute. Thirtymicroliter purified conjugate sample (or Factor VIII as control) werediluted with 30 ∞L of 2 mM HCl and placed in an HPLC vial with 200 μL.The following gradient was used: for time zero, 0% of the mobile phasecontained solution B; for time zero to two minutes, 0% of the mobilephase contained solution B; for time two minutes to 2.5 minutes, 27% ofthe mobile phase contained solution B; for time 2.5 minutes to 8minutes, 27% of the mobile phase contained solution B; for time 8minutes to 8.5 minutes, 70% of the mobile phase contained solution B;for time 8.5 minutes to 14 minutes, 70% of the mobile phase containedsolution B. For each injection, 30 μL of sample or control were used. Inthe chromatogram at 280 nm, peaks will correspond to the following:native Factor VIII at about 11 minutes and conjugated Factor VIII wasearlier. A typical chromatogram prepared in accordance with thisprocedure is provided as FIG. 2B.

The purified conjugate sample was analyzed by SDS-PAGE by allowing a3-8% TRIS-acetate gel (Invitrogen Corporation, Carlsbad Calif.) warmingto room temperature, wherein a standard curve of polymeric reagent B in2 mM HCL at concentrations of 0.001%, 0.01% and 0.1% of polymericreagent B (w/v). The standard was prepared by placing 10 μL of HiMarkmolecular weight marker (Invitrogen Corporation, Carlsbad Calif.) intolane 1. Purified conjugate sample or control (Factor VIII) (10 μLvolumn) were each individually diluted with 30 μL of 2 mM HCl, wherein30 microliters of each HCl diluted sample or control was combined with10 μL of 4× LDS Sample Buffer (Invitrogen Corporation, Carlsbad Calif.),wherein 25 μL of the solution was then transferred to the designatedwell. Immediately, the gel was placed in the gel apparatus and was runfor 60 minutes at 150 volts. Following completion of the run, the gelwas removed from the gel apparatus and rinsed in deionized water. Thegel was then stained with a barium iodine stain (performed by: adding 15mL 0.1 M perchloric acid to the gel followed by a five minute incubationperiod; followed by addition to the gel of 5 mL of 5% barium chloridethen 2 mL of iodine followed by a five minute incubation period)followed by rinsing with deionized water. Five minutes after the gel wasrinsed with deionized water, the gel was analyzed with a Kodak Gel LogicScanner system (Eastman Kodak Company, New Haven Conn.), whereinunreacted polymeric reagent B was identified. After scanning, anyremaining water was poured off the gel and 50 mL of Pierce ImperialStain (Pierce Biotechnology, Inc., Rockford Ill.) was added to the gel.Following incubation at room temperature for thirty minutes, the gel wasrinsed with deionized water and allowed to stand for one hour in 200 mLof deionized water. During the hour period, several changes of waterwere completed. After the hour, the gel was analyzed with a Kodak GelLogic Scanner system (Eastman Kodak Company, New Haven, Conn.).

EXAMPLE 2A1 Preparation of FVIII Conjugate (20,000 Da Total PolymerWeight Average Molecular Weight) (“Lys 20K br Long—Resynthesized”)

The synthetic procedure of Example 2A was repeated. Upon carrying outthe procedure again, it was noted that some differences in the polymerto Factor VIII ratio was observed between the resynthesized conjugatesand those of Example 2A, which might be explained by the use ofdifferent analytical methods. As investigated by barium-iodine staining,however, no free polymeric reagent B remained in any sample solution.

EXAMPLE 2B Preparation of FVIII Conjugate (40,000 Da Total PolymerWeight Average Molecular Weight) (“Lys 40K br Long”)

The basic procedure of Example 1A was repeated except that polymericreagent B having a total polymer weight average molecular weight ofabout 40,000 Da was used instead of about 20,000 Da.

EXAMPLE 2B1 Preparation of FVIII Conjugate (40,000 Da Total PolymerWeight Average Molecular Weight) (“Lys 40K br Long—Resynthesized”)

The synthetic procedure of Example 2B was repeated. Upon carrying outthe procedure again, it was noted that some differences in the polymerto Factor VIII ratio was observed between the resynthesized conjugatesand those of Example 2A, which might be explained by the use ofdifferent analytical methods. As investigated by barium-iodine staining,however, no free polymeric reagent B remained in any sample solution.

EXAMPLE 2C Preparation of FVIII Conjugate (60,000 Da Total PolymerWeight Average Molecular Weight) (“Lys 60K br Long”)

The basic procedure of Example 2A was repeated except that polymericreagent B having a total polymer weight average molecular weight ofabout 60,000 Da was used instead of about 20,000 Da.

EXAMPLE 3A Preparation of vWF Conjugate (20,000 Da Total Polymer WeightAverage Molecular Weight) (“Lys 20K br Short”)

-   -   (wherein VWF is a residue of von Willebrand Factor)

(175 mL) of von Willebrand Factor (“VWF”) solution (0.344 mg/mL in 20 mMHEPES, 150 mM NaCl, 0.5% Sucrose, pH 7.4) was allowed to thaw to roomtemperature. A 175 molar ratio (relative to VWF) of polymeric reagent A(766.3 mg) having a total polymer weight average molecular weight (i.e.,the sum of the weight average molecular weight of each polymer “arm”) ofabout 20,000 Da, which was freshly dissolved in 7.7 mL of 2 mM HCl, wasslowly pipetted into the VWF solution. The mixture was allowed to shakegently on a shaker for two hours at room temperature. The reaction wasquenched by addition of 1.8 mL of 1 M glycine in water, which wasallowed to shake gently on a shaker at room temperature for anotherthree hours. The solution was diluted by slow addition of 175 mL of 20mM MES Buffer at pH 6.10 with 0.5 wt % sucrose. The solution was mixedwell by gentle swirling, and then was stored at 4° C. overnight. Theunbound polymeric reagent A in the solution was then removed by ionexchange chromatography. See the chromatogram below. The resultingconjugate was characterized by SDS-PAGEs. The chromatogram followinganion exchange chromatography is provided in FIG. 3A. FIGS. 3B and 3Cshows the gels following SDS-PAGE analysis under reduced and non-reducedconditions, respectively.

EXAMPLE 3B Preparation of vWF Conjugate (40,000 Da Total Polymer WeightAverage Molecular Weight) (“Lys 40K br Short”)

An aliquot (175 mL) of von Willebrand Factor (“VWF”) solution (60.2 mgprotein content) was allowed to thaw to room temperature. A 135 molarratio (relative to VWF) of polymeric reagent A (1.374 g) having a totalpolymer weight average molecular weight (i.e., the sum of the weightaverage molecular weight of each polymer “arm”) of about 40,000 Da,which was freshly dissolved in 13.7 mL of 2 mM HCl, was slowly pipettedinto the VWF solution. The mixture was allowed to shake gently on ashaker for three hours at room temperature. The reaction was quenched byaddition of 945 μL of 2 M glycine in water, which was allowed to shakegently on a shaker at room temperature for another three hours. Thesolution was diluted by slow addition of 175 mL of 20 mM MES Buffer atpH 6.10 with 0.5 wt % sucrose. The solution was mixed well by gentleswirling, and then was stored at 4° C. overnight. The unbound polymericreagent A in the solution was then removed by ion exchangechromatography. See FIG. 4A for the corresponding chromatogram.

EXAMPLE 3C Preparation of vWF Conjugate (60,000 Da Total Polymer WeightAverage Molecular Weight) (“Lys 60K br Short”)

An aliquot (175 mL) of von Willebrand Factor (“VWF”) solution (60.2 mgprotein content) was allowed to thaw to room temperature. A 150 molarratio (relative to VWF) of polymeric reagent A (2.406 g) having a totalpolymer weight average molecular weight (i.e., the sum of the weightaverage molecular weight of each polymer “arm”) of about 60,000 Da,which was freshly dissolved in 13.7 mL of 2 mM HCl, was slowly pipettedinto the VWF solution. The mixture was allowed to shake gently on ashaker for three hours at room temperature (22° C.). The reaction wasquenched by addition of 875 μL of 2 M glycine in water, which wasallowed to shake gently on a shaker at room temperature for anotherthree hours. The solution was diluted by slow addition of 175 mL of 20mM MES Buffer at pH 6.10 with 0.5 wt % sucrose. The solution was mixedwell by gentle swirling, and then was stored at 4° C. overnight. Thefree PEG in the solution was then removed by ion exchangechromatography. See FIG. 4B for the corresponding chromatogram.

EXAMPLE 4A Preparation of Factor VIII Conjugate (20,000 Da Total PolymerWeight Average Molecular Weight) (“Lys 20K br Short”)

-   -   (wherein FVIII is a residue of Factor VIII)

Factor VIII protein solution (3.23 mg/mL protein concentration) wasquickly thawed (using a warm water bath at room temperature for fiveminutes) and, using 165 μL of the warmed FVIII protein solution wasplaced intpa 1 mL microcentrifuge tube. The microcentrifuge tube wasplaced in standard ice bath (not dry ice as solution should not freeze),thereby forming a chilled Factor VIII protein solution.

A 70 molar ratio (relative to Factor VIII) of polymeric reagent A havinga total polymer weight average molecular weight (i.e., the sum of theweight average molecular weight of each polymer “arm”) of about 20,000Da was placed into a 1 mL microcentrifuge tube. The weighed polymericreagent A was suspended 2 mM HCl to form a polymeric reagent A solution.After ensuring that the polymeric reagent A was dissolved (achieved byvortexing the solution for five seconds followed by centrifuging for tenseconds), all of the polymeric reagent A solution was added to thechilled Factor VIII protein solution, the resulting mixture was placedon a rocker plate at room temperature for one hour. At the end of onehour, 18.8 μL of a 50 mM glycine solution was added to thereby form aglycine-containing PEGylation reaction mixture. The glycine-containingPEGylation reaction mixture was rocked for twenty minutes at roomtemperature on a rocker plate.

To remove salt within the glycine-containing PEGylation reactionmixture, a mL HiTrap DeSalt column was pre-equilibrated with 20 mM MOPS,10 mM CaCl₂, 0.1% Tween 80, pH 6.5. Once equilibrated, the reaction wasdiluted with Milli-Q water (Millipore Corporation, Billerica, Mass.) toa final volume of 1 mL. The entire volume was then loaded onto thecolumn and fractions were collected and pooled. Protein-containingfractions were immediately placed in standard ice bath. A typicalchromatogram prepared in accordance with this procedure is providedbelow.

To purify the conjugate within the desalted glycine-containingPEGylation reaction mixture (to remove unconjugate PEG species), theconjugate was purified by cation exchange chromatography on an ÄKTABasic System. The column used was a 5 mL HiTrap Q HP column (regeneratedwith 20 mM MOPS, 10 mM CaCl₂, 0.1% Tween 80, pH 6.5+1 M NaCl andpre-equilibrated with 20 mM MOPS, 10 mM CaCL₂, 0.1% Tween 80, pH 6.5.The desalted glycine-containing PEGylation reaction mixture was loadedonto the column and purification was carried out with a step gradientfom 0-50% mM MOPS, 10 mM CaCl2, 0.1% Tween 80, pH 6.5+1 M NaCl. Thefractions were collected, pooled and stored in a container that wasplaced immediated in standard ice bath. See the chromatogram provided asFIG. 5.

The purified conjugate was analyzed by SDS-PAGE by allowing a 3-8%TRIS-acetate gel (Invitrogen Corporation, Carlsbad Calif.) warming toroom temperature, wherein a standard curve of of polymeric reagent B in2 mM HCL at concentrations of 0.001%, 0.01% and 0.1% of polymericreagent B (w/v). The standard was prepared by placing 10 μL of HiMarkmolecular weight marker (Invitrogen Corporation, Carlsbad Calif.) intolane 1. Purified conjugate sample or control were each individuallydiluted with 30 μL of 2 mM HCl, wherein 30 microliteres of each HCldiluted sample or control was combined with 10 μL of 4× LDS SampleBuffer (Invitrogen Corporation, Carlsbad Calif.), wherein 25 μL of thesolution was then transferred to the designated well. Immediately, thegel was placed in the gel apparatus and was run for 60 minutes at 150volts. Following completion of the run, the gel was removed from the gelapparatus and rinsed in deionized water. The gel was then stained with abarium iodine stain (performed by: adding 15 mL 0.1 M perchloric acid tothe gel followed by a five minute incubation period; followed byaddition to the gel of 5 mL of 5% barium chloride then 2 mL of iodinefollowed by a five minute incubation period) followed by rinsing withdeionized water. Five minutes after the gel was rinsed with deionizedwater, the gel was analyzed with a Kodak Gel Scanner (Eastman KodakCompany, New Haven Conn.). See FIG. 6A.

After scanning, any remaining water was poured off the gel and 50 mL ofGel Code Blue (Invitrogen Corporation, Carlsbad Calif.) was added to thegel. Following incubation at room temperature for thirty minutes, thegel was rinsed with deionized water and allowed to stand for one hour in200 mL of deionized water. During the hour period, several changes ofwater were completed. After the hour, the gel was analyzed with a KodakGel Scanner (Eastman Kodak Company, New Haven Conn.), wherein uncongatedFactor VIII was identified. See FIG. 6B.

EXAMPLE 4B Preparation of Factor VIII Conjugate (40,000 Da Total PolymerWeight Average Molecular Weight) (“Lys 40K br Short”)

The basic procedure of Example 4A was repeated except that polymericreagent A having a total polymer weight average molecular weight ofabout 40,000 Da was used instead of about 20,000 Da.

EXAMPLE 4B1 Preparation of Factor VIII Conjugate (40,000 Da TotalPolymer Weight Average Molecular Weight) (“Lys 40K brShort—Resynthesized”)

The basic procedure of Example 4A was repeated except that: (a)polymeric reagent A having a total polymer weight average molecularweight of about 40,000 Da was used instead of about 20,000 Da; and (b) amolar excess of 150 of polymeric reagent A relative to Factor VIII wasused for the conjugation step, wherein 5 mg of polymeric reagent A wasplaced into a clean 1 mL microcentrifuge tube and was dissolved in 50 μLof 2 mM HCl. Vortex the solution for 5 seconds, then centrifuge for 10seconds to completely dissolve the PEG. Add all 50 μL of PEG solution tothe chilled FVIII and place on rocker plate at room temperature for 1hour. Quench with 21.5 μL of 50 mM Glycine. Continue rocking for 20minutes at room temperature.

EXAMPLE 4C Preparation of Factor VIII Conjugate (60,000 Da Total PolymerWeight Average Molecular Weight) (“Lys 60K br Short”)

The basic procedure of Example 4A was repeated except that polymericreagent A having a total polymer weight average molecular weight ofabout 60,000 Da was used instead of about 20,000 Da.

EXAMPLE 5 In Vitro and In Vivo Experiments of PEGylated rVWF

Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) wasperformed under reducing conditions as described in this Example 5followed by silver staining (FIG. 7, Panel A) and Coomassie staining(FIG. 7, Panel B). Under reducing conditions mature rVWF appeared as aprominent single band (monomer) with a MW of 260 kDa with some minorbands down to 150 kDa. When the gels were immunoblotted with apolyclonal man VWF antibody, as demonstrated in FIG. 7, Panel C, rVWFmonomers show MWs apparently higher than 280 kD due to the use ofdifferent MW standards. Native rVWF 133P1 appeared as a single VWFmonomer in the anti-VWF immunoblot with some minor, non-relevantdegradation bands. Multimer composition was investigated by non-reducingagarose gel electrophoresis using a high resolution gel to demonstratethe integrity of the multimeric structure and to confirm that nosatellite or other degradation bands had occurred (FIG. 7, Panel D).Analytical data of Native rVWF 133P1 is provided in Table 1.

TABLE 1 Analytical Data of Native rVWF 133P1 Starting material rVWF133P1 Protein Bradford (mg/ml) 0.344 VWF:Ag (IU/ml) 54.6 VWF:Ag/totalprotein (IU/mg) 158.7 VWF:RCo (IU/ml) 24.3 VWF:RCo (IU/IU Ag) 0.45VWF:CB (U/ml) 63.5 VWF:FVIIIB capacity (%) 84 VWF:FVIIIB (U/ml) 46VWF:FVIIIB affinity; KD (M) 5.49E−10

Releasable rVWF conjugates: PEG-rVWF conjugates with releasable bondsvia the amino groups of the lysine residues of rVWF were prepared inaccordance with Examples 1A, 1B, 1C, 3A, 3B and 3C. The PEG-rVWFconjugates had total PEG molecular weights of about 20K, 40K or 60K.Table 2 summarizes the PEGylation degree of the conjugates.

TABLE 2 PEGylation degree of the releasable PEG-rVWF preparationsPEG/monomer Protein SDS- Material Name (mg/ml) PAGE* Ferrothiocyanate**HPLC Free PEG Lys 20K br short low 0.304 1-2 — 3.7 <0.002% Lys 20K brlong low 0.450 <2 5.2 4.0 <0.002% Lys 40K br short low 0.228 <2  2.183.2 <0.002% Lys 40K br long low 0.315 1 — 1.9 <0.001% Lys 60K br shortlow 0.216 <2 1.6 4.2 <0.005% Lys 60K br long low 0.410 1 — 3.2<0.00625%  The protein content of the PEGylated rVWF was determinedusing the Bradford assay with unmodified rVWF 133P1 as standard. Theremaining free PEG was determined by barium iodide staining of thenon-reducing SDS-PAGE. *The number of PEGs per molecule was counted byanalyzing the Coomassie stained reducing SDS-PAGE followed bygel-scanning analysis. **The number of PEGs per molecule was determinedwith the pronase/ammonium ferrothiocyanate method.

Some differences in the PEG to VWF monomer ratio determined were shownwith the different methods applied. The HPLC method, which does notrequire a PEG extraction step, gave an average higher number ofPEG/monomer than that given by the SDS-PAGE method. Thus, the originaltarget of a low PEGylation degree of 1-2 PEG/VWF monomer(“monoPEGylation”) was exceeded in all preparations except in the Lys40K br long low conjugate. As investigated by barium iodide staining, nofree PEG remained in the sample solution.

The protein content of the samples was measured according to theprinciple described by Bradford using the Protein Assay Dye ReagentConcentrate from Bio-Rad Laboratories (Hercules, Calif., USA). Bradford(1976) Anal. Biochem. 72:248-254. The microassay procedure was performedaccording to the manufacturer's instructions and calibrated using acertified human serum preparation (Qualitrol HS-N, DiaSys Diagnostics,Holzheim, Germany; distributed by VWR, Darmstadt, Germany), obtaining acalibration range of 20 to 1.8 μg protein/ml. Pre-dilution ofconcentrated samples as well as sample dilutions were prepared with 0.9%NaCl solution.

Determination of VWF:AG: Two different VWF:Ag assays were used duringthe experiments: SIMIT and sandwich ELISA.

A single incubation multilayer immune technique (SIMIT) for the in vitrocharacterization of the conjugates was one analytic technique used. Thedouble sandwich VWF:Ag ELISA was set up with a commercially availableantibody combination of a polyclonal rabbit anti-human VWF:antibody(A-082) and a peroxidase-labeled polyclonal rabbit anti-human VWFantibody (P-0226; both obtained from Dakopatts, Glostrup, Denmark) usingthe single incubation multilayer immune technique SIMIT. Wells ofmicrotiter plates (NUNC Maxisorb F96; obtained from VWR) were coatedwith 1 μg antihuman VWF:antibody diluted in coat buffer (100 mM sodiumcarbonate, 100 mM sodium hydrogen carbonate, adjusted to pH 9.5 withHCl). Phosphate-buffered saline containing Tween-20 (PBST) used aswashing buffer was composed of 137 mM NaCl, 2.7 mM KCl, 1.5 mM potassiumdihydrogen phosphate, 7 mM disodium hydrogenphosphate dihydrate and 0.5ml Tween-20 (Bio-Rad, EIA grade). For the dilution of samples and theantibody conjugate, 0.1% non-fat dry milk and 2 mM benzamidine was addedto the washing buffer. All incubations were done at room temperature.Peroxidase activity was detected by using tetramethyl-benzidine (TMB) assubstrate. The developed color intensity was measured with an ELISAreader at 450 nm. A normal reference plasma calibrated against theactual WHO standard was used for the construction of a calibrationcurve. A dilution series consisting of the six geometric 1+1 dilutions1/100-1/3200 was prepared and analyzed in duplicates on each singleplate obtaining a VWF:Ag concentration range of 0.01-0.0006 IU/ml.Samples were diluted at least 1+1 and five further 1+1 dilutions wereanalyzed in duplicates. The assay blank was also run in duplicates. Forthe data evaluation, a linear regression curve was calculated betweenthe logarithms of both blank-corrected optical densities (ODs) measuredand the known VWF:Ag concentrations of the six calibrators. Sample ODswere extrapolated on this curve only when they were within the rangedefined by the calibration curve and results were reported in IU/ml.

Sandwich ELISA for in vitro release experiments and for analysis of exvivo plasma samples from the pharmacokinetic studies was also used.VWF:Ag was determined with a sandwich ELISA. Wells of microplates(Nunc-immuno 96-microwell plates, Maxisorp, Nunc, Roskilde, Denmark)were coated overnight with 1 μg/well polyclonal anti-VWF antibody(A-082, Dako, Glostrup, Denmark) in 50 mM sodium bicarbonate buffer, pH9.6. Plates were then washed with washing buffer (20 mM Tris, 140 mMNaCl, 0.1% Tween-20, pH 7.4), and diluted samples in the range from 0.02to 0.001 IU/ml VWF:Ag [in washing buffer+0.3% bovine serum albumin(BSA)] were incubated in the wells for 2 hours at 25-32° C., followed bywashing and incubation with polyclonal anti-human VWF conjugated withhorseradish peroxidase (P-0226, Dako, Glostrup, Denmark) in washingbuffer +0.3% BSA. After a washing step, 0.4 mg/ml ofortho-phenylenediamine (P1063, Sigma, St. Louis, Mo., USA) inphosphate-citric acid buffer was added. Color development was stoppedwith 3 M sulfuric acid. Absorbance of wells was read in a microplatereader at 492 nm. Absorbance is directly proportional to VWF content inthe sample. The VWF:Ag concentration in the samples was calculatedrelative to a human plasma reference preparation (SSC/ISTH secondarycoagulation standard, #2).

Determination of VWF:RCo activity: VWF:RCo activities were measured withthe BCS (Behring Coagulation System) analyzer (Dade Behring, Marburg,Germany) according to the instructions of the manufacturer by use of alyophilized von Willebrand reagent containing stabilized platelets andristocetin A (Dade Behring). The VWF (ristocetin cofactor) from thesample causes agglutination of the stabilized platelets in the presenceof ristocetin. The resulting agglutination decreases the turbidity ofthe reaction suspension. The change in absorbance measured by the BCSanalyzer at 570 nm is proportional to the sample's ristocetin cofactoractivity. The ristocetin cofactor activity of the sample is quantifiedby means of a reference curve generated with Standard Human Plasma (DadeBehring) and reported in IU VWF:RCo/ml.

Determination of the collagen-binding activity: The VWF:CB activity wasdetermined with a commercially available ELISA (Technozym VWF:CBA,Technoclone, Vienna, Austria) according to the instructions of themanufacturer. The precoated ELISA test strips with the immobilized humancollagen type III were incubated with the sample solution.Collagen-bound VWF was detected by adding a peroxidase-conjugatedpolyclonal anti-VWF antibody. The VWF:CB activity of the sample wasquantified by means of a reference curve generated with normal humanplasma provided with the test-kit and expressed in U/ml VWF:CB.

Determination of VWF-FVIII-binding capacity by an ELISA chromogenicassay (ECA): The VWF-FVIII interaction was determined by an ECA based onthe assay described by Bendetowicz et al. See Bendetowicz et al. (1998)Blood 92(2):529-538. A commercially available polyclonal rabbitanti-human VWF antibody A082 (Dako, Glostrup, Denmark) was immobilizedto the microtiter wells. Phosphate-buffered saline (PBS; 6.5 mM disodiumhydrogenphosphate dihydrate, 1.5 mM potassium dihydrogen phosphate, 140mM NaCl, pH 7.2) containing 0.05% Tween-20 was used as washing buffer.For sample dilution and as blocking solution, 0.1% non-fat dry milk(Bio-Rad, Hercules, Calif., USA) was added to the PBS-Tween buffer. Aconstant amount of rFVIII [0.2 IU/ml FVIII chromogenic activity(FVIII:C)] was mixed with the diluted VWF-containing sample (VWF:Agconcentration range 0.156 to 10 mIU/ml) in separate tubes and incubatedat 37° C. for 25 minutes. The rFVIII source was a frozen bulk derivedfrom the ADVATE brand of Factor VIII (Baxter Healthcare). The rFVIIIbulk had a chromogenic FVIII activity of 4046 IU/ml, and contained lessthen 5 μg (˜0.5 units) VWF:Ag/1000 IU of rFVIII. The rFVIII product wasstored frozen in aliquots below −60° C. and thawed immediately beforethe assay.

This VWF-FVIII complex was transferred to the blocked microtiter plateand incubated for 60 minutes at room temperature. Unbound FVIII wasremoved by a subsequent washing step with washing buffer. Bound FVIIIwas quantified by a commercially available FVIII chromogenic assay(Technochrom FVIII:C reagent kit, Technoclone, Vienna, Austria), inwhich the reagent contains minute amounts of thrombin, FIXa,phospholipids and FX. The principle of this assay is that FVIII isactivated by thrombin and thus released from VWF and subsequently formsa complex with phospholipid, FIXa and calcium ions. This complexactivates FX to FXa, which in turn cleaves the chromogenic substrate,resulting in a color reaction measured in an ELISA reader (Benchmark,Bio-Rad, Hercules, Calif., USA) at 405 nm using the kinetic mode at 37°C. All samples were serially diluted and analyzed in duplicates. Theblank corrected optical densities received (in mOD/min) were plottedagainst the VWF:Ag concentrations in logarithmic scale. TheFVIII-binding activity of the sample was calculated from a fittedreference curve constructed from a normal reference plasma assuming that1 IU of VWF:Ag has 1 U of VWF:FVIIIB activity.

For the applied concentration range from 0.156 to 10 mIU/ml VWF:Ag, theendogenous FVIII, which is bound to VWF of the normal plasma, had noinfluence on the measurement. The VWF:FVIIIB activity of the samples wasexpressed in U/ml, as read from the reference curve and theFVIII-binding capacity was calculated as a percent of the VWF:Agmeasured in the sample.

Measurement of VWF-FVIII affinity by surface plasmon resonancetechnology: Unmodified and PEGylated VWF were immobilized on the flowcells of a CM5 sensor chip of a Biacore 3000 (Biacore AG, Uppsala,Sweden) apparatus to a constant level according to the instructions ofthe manufacturer. A series of dilutions of FVIII samples were thenapplied to the chip using the “kinject” mode, allowing 3 minutes for theassociation and 10 minutes for the dissociation of FVIII. After each ofthese cycles, FVIII was removed from the chip (“regeneration”) and theexperiment was repeated with a new FVIII sample.

Determination of the FVIII-binding capacity of PEG-rVWF in the presenceof native rVWF under flow conditions: A constant amount of rVWF wasimmobilized on the flow cells of a CM5 sensor chip of a Biacore 3000(Biacore AG, Uppsala, Sweden). Different amounts of rVWF were incubatedwith 5 IU/ml rFVIII at 37° C. for 5 minutes to form a complex and theninjected into the flow cells with the immobilized rVWF. The amount offree FVIII bound to the immobilized rVWF was calculated from a referencecurve, established by injecting rFVIII in the absence of rVWF in therange of 0.1 to 5 IU/ml. The rFVIII remaining in the complex wascalculated and expressed as a percent of the added rFVIII bound in theabsence of rVWF.

Measurement of susceptibility for VWF cleaving protease (ADAMTS13):Susceptibility of rVWF to ADAMTS13 was investigated by incubating theconjugates with increasing concentrations of preactivated ADAMTS13 underdenaturing conditions to unfold the VWF. The degradation of VWF wasmeasured by collagen-binding (VWF:CB) activity, which depends on themultimeric size of VWF, before and 4 hours after the incubation. Thedegradation of the multimer numbers and formation of the specificsatellite bands were visualized by multimer analysis.

For the degradation of rVWF, normal human plasma (George KingBio-Medical, Overland Parks, Kans., USA) was used, as the ADAMTS13source. ADAMTS13 in the dilutions of the plasma were activated withBaCl₂ for 30 minutes at 37° C. in the presence of 5 mM Tris, 1.5 M urea,pH 8.0 and mixed with constant amounts of rVWF (prediluted with 5 mMTris, 1.5 M urea, pH 8.0) and further incubated at 37° C. for 4 hours.The incubation mixtures contained 6 μg/ml of native or PEGylated rVWFconjugates and 1 to 33 mU/ml of ADAMTS13. The reaction was stopped bythe addition of Na₂SO₄ and the incubation mixtures were subsequentlycentrifuged for 5 minutes at 2500 g and the supernatant was used forfurther analysis.

Collagen-binding activity (VWF:CB) was determined. High-binding 96-wellELISA plates (Costar 3590, Corning Incorporated, NY, USA) were precoatedwith 100 μl of 1.5 μg/ml human collagen type III (Southern BiotechnologyAssociates, Inc., Birmingham, USA) in 6.5 mM di-sodium hydrogenphosphate dihydrate, 1.5 mM kalium dihydrogen phosphate, 140 mM NaCl, pH7.2 (PBS) overnight at 4° C. and subsequently blocked with 200 μl of“Super Block Blocking Buffer in PBS” (Pierce, Rockford, Ill., USA) for30 minutes at room temperature. The centrifuged digestion mixtures werediluted 1/5 with PBS containing 0.05% Tween-20 and 10% of the blockingsolution and 100 μl of these dilutions were added to the blocked wells.After incubation for 2 hours at room temperature, the plates werefurther incubated for one hour with 100 μl of a solution of polyclonalhorse-radish peroxidase-conjugated anti-human VWF antibody (P-0226,Dako, Glostrup, Denmark) diluted 1/10000 in PBS buffer, pH 7.2containing 0.05% Tween-20 and 10% of the blocking solution. Between eachstep, the microtiter wells were washed three times with 250 μl PBScontaining 0.05% Tween-20. The color reaction was achieved by additionof 100 μl of “ImmunoPure TMB Substrate” (Pierce, Rockford, Ill., USA),and after 5 minutes incubation the reaction was terminated by theaddition of 100 μl 1 N H₂SO₄. The absorbance was read at 450 nm using anELISA reader 680 (Bio-Rad Laboratories, Hercules, Cailf., USA). As anegative control, physiological saline was used instead of normal plasmawith the same procedure. Samples with 0.017 U/ml ADAMTS13 were subjectedto low- and high-resolution multimer analysis as described below under“VWF multimer analysis.”

SDS-PAGE and silver staining: VWF samples (20 mIU, equal to 0.2 μgprotein per lane) were applied to gradient (3-8%) Tris-acetate gels andelectrophoresis was done under reducing conditions, followed by silverstaining, as described by the manufacturer (Bio-Rad). As molecularweight standard the Precision Plus Protein All Blue standard was used(250-10 kDa, Bio-Rad, Hercules, Calif., USA).

SDS-PAGE and Coomassie staining: VWF samples (100 mIU, equal to 1 μgprotein per lane) were applied to gradient (3-8%) Tris-acetate gels andelectrophoresis was done under reducing conditions, followed byCoomassie staining, as described by the manufacturer (Bio-Rad, Hercules,Calif., USA). The Precision Plus Protein All Blue standard was used(250-10 kDa/Bio-Rad, Hercules, Calif., USA) as the molecular weightstandard.

SDS-PAGE and immunoblot for VWF VWF samples (0.55 mIU equal to 5.5 ngprotein per lane) were applied to gradient (3-8%) Trisacetate gels andelectrophoresis was done under reducing conditions, followed by standardblotting procedures onto a polyvinylidene difluoride (PVDF) membrane. Tovisualize the VWF bands, a polyclonal rabbit anti-human VWF antibody(A-082, Dako, Glostrup, Denmark) was used as primary antibody. Analkaline phosphatase (ALP)-labeled goat anti-rabbit IgG was applied as asecondary antibody (Bethyl Laboratories Inc., Montgomery, Tex., USA).The blots were developed with the ALP Conjugate Substrate Kit (Bio-Rad,Hercules, Calif., USA). A full range rainbow marker (250-10 kDa,GE-Healthcare, Little Chalfont, Buckinghamshire, UK) was used as themolecular weight standard.

SDS-PAGE and immunoblot for PEG VWF samples (5.5 mIU equal to 55 ngprotein per lane) were applied to gradient (3-8%) Tris-acetate gels andelectrophoresis was done under reducing conditions, followed by standardblotting procedures onto a PVDF membrane. To visualize the PEG,polyclonal rabbit anti-human PEG antibody was used as primary antibody.The anti-PEG antibody was raised in rabbits by immunization with aPEGylated protein. The IgG fraction of the rabbit serum was purified byaffinity chromatography on Protein G Sepharose 4B (GE-Healthcare,Uppsala, Sweden) followed by specific negative immunabsorption. AnALP-labeled goat anti-rabbit IgG (Bethyl Laboratories Inc., Montgomery,Tex., USA) was applied as a secondary antibody. The blots were developedwith the ALP Conjugate Substrate Kit (Bio-Rad, Hercules, Calif., USA). Afull range rainbow marker (250-10 kDa, GE-Healthcare, Little Chalfont,Buckinghamshire, UK) was used as the molecular weight standard.

VWF multimer analysis The size distribution of the rVWF preparationswere analyzed by high-density horizontal SDS agarose gel electrophoresisusing high-resolution (2.5-2.7% agarose) conditions. Samples werediluted to the same concentration in the range of 0.3-1.0 IU/ml VWF:Agand incubated with Tris-EDTA-SDS buffer. The multimers were separatedunder non-reducing conditions on an agarose gel.

VWF multimers and the distribution of PEG on the VWF multimers wereeither visualized in the gel by immunostaining with a polyclonal rabbitanti-human VWF antibody (A-082, Dako, Glostrup, Denmark) or with apolyclonal rabbit anti-PEG antibody after electroblotting to aPVDF-membrane, followed by ALP-conjugated goat anti-rabbit IgG H+L(Jackson Immuno Research, Soham, Cambridgeshire, UK) using the ALPConjugate Substrate Kit (Bio-Rad, Hercules, Calif., USA).

As a hemophilia model, FVIII-knockout mice [Lawler et al. (1995) Nat.Genet. 10(1):119-121] were used. The mice suffered from severehemophilia A (FVIII<0.01 IU/ml) but had normal levels of VWF(approximately 0.15 IU/ml relative to human VWF reference), mimickinghuman hemophilia A.

Application of VWF and FVIII: Recombinant FVIII (214 IU FVIII/ml) fromBaxter was used in all experiments in this example and co-injected withrVWF. The freeze-dried final containers were stored at 2-8° C. andreconstituted before use. The dissolved rFVIII and non-PEGylated orPEGylated rVWF were mixed with 20 mM Hepes, 150 mM NaCl, 3.2% mannitol,0.8% trehalose, 2.5 mM CaCl₂, 1% human albumin, pH 7.4 buffer to achieveappropriate concentrations for infusion. The mixtures were aliquoted,frozen at −20° C. and thawed just before the applications. Target dosewas 200 IU/ml FVIII:C and 1.6 to 2.1 mg/kg VWF. The concentrations weremeasured again from the thawed samples and the applied doses werecalculated. Doses are given in the Figures legends. 10 ml/kg bodyweightwere injected via the tail vein and groups of 5-6 mice were bled byheart puncture after 5 minutes, 3 hours, 6, 9, 16 and 24 hours, and ifnecessary, after 32 and 42 hours. Nine volumes of blood were mixed with1 volume of 3.8% sodium citrate, and immediately centrifuged at 3000 gfor 10 minutes. The supernatant was again centrifuged at 3000 g for 5minutes, plasma was separated, frozen in aliquots and stored below −60°C. for analysis.

Determination of FVIII activity in mouse plasma: FVIII activity wasdetermined with a chromogenic method following the assay principle asset forth above with respect to the determination of VWF-FVIII bindingcapacity by an ELISA chromogenic assay. The time course of thepara-nitroaniline (pNA) released from the substrate was measured with amicroplate reader at 405 nm using the kinetic mode. The slope of thereaction is proportional to the FVIII concentration in the sample. TheFVIII concentration in the samples was calculated relative to a humanplasma reference preparation, calibrated against the WHO plasmareference (5^(th) IS for FVIII and VWF in human plasma, NIBSC #02/150)and expressed in IU/ml.

Determination of VWF antigen in mouse plasma: VWF:Ag was determined withthe sandwich ELISA described above with respect to Sandwich ELISA for invitro release experiments and for analysis fo ex vivo plasma samplesfrom the pharmacokinetic studies. The VWF:Ag concentration of thesamples was calculated relative to a human plasma reference preparation(SSC/ISTH secondary coagulation standard, #2). The baseline-level ofmouse VWF was subtracted. The quantification limit of the assay in mouseplasma was 0.03 IU/ml of VWF:Ag.

Calculation of the circulating half-life parameters of human VWF andFVIII: For analyzing FVIII levels, the concentrations for t₀=0 hours wasset to zero as FVIII deficient mice were studied. For analyzing VWF:Aglevels, the concentration for t₀=0 was set to zero and the arithmeticmean concentration of untreated mice was subtracted from meanconcentrations at subsequent time points. FVIII levels over time weresummarized using pharmacokinetic parameters AUC from zero to 24 hours,terminal elimination rate and mean residence time. VWF:Ag levels overtime were summarized by the pharmacokinetic parameter AUC from zero to24 hours.

Area under the concentration vs. time curve (AUC) from 0 to 24 hours:The area under the concentration vs. time curve (AUC) from 0 to 24 hourswas calculated by the linear trapezoidal rule using the arithmetic meansof the concentrations observed at individual time points. It was assumedthat there exists a linear relationship between dose and AUC. Under thisassumption, the AUCs for different items were adjusted for dose in caseof different doses administered. Dose adjustment was performed bydividing the calculated AUC by the dose per kg body mass administered.

Terminal Elimination Rate: The terminal elimination rate (λ) wasestimated using the arithmetic mean of the natural logarithms ofindividual concentrations at the last three time points modified with abias correction. See Wolfsegger et al. (2005) J. Pharmacokinetic.Pharmacodyn. 32(5-6):757-766.

Mean Residence Time: Mean residence time (MRT) was calculated asAUMC_(0-infinity) divided by AUC_(0-infinity). AUMC_(0-infinity) andAUC_(0-infinity) were calculated by the linear trapezoidal rule usingthe arithmetic means of the concentrations observed for different timepoints plus a three-point tail area correction. The tail area correctionwas calculated by log-linear fitting on the arithmetic means observed atthe last three time points per item.

Results

Functional parameters of PEG-rVWF: The different biological functions ofVWF were characterized by different parameters. VWF:CB, VWF:RCo andVWF:FVIIIB were measured to characterize the integrity of the collagenand platelet-binding site, required for the VWF-mediated plateletadhesion, one of the first steps of hemostasis. The VWF:FVIIIB capacityand affinity describes the availability of the FVIII-binding sites,needed for the chaperon function of VWF. Table 3 summarizes the measuredvalues, while the calculated ratios and specific activities aresummarized in Table 4.

To investigate the possible effect of the conjugation with thereleasable PEG reagents, a mock preparation (control) was alsomanufactured which ran through the whole process but was not PEGylated.This preparation had similar activities to the controls, which confirmedthat the process had no negative effect on rVWF.

Because all parameters were similarly affected, the VWF:FVIIIB capacity,which was expressed as a percent of the VWF:Ag level, only marginallydecreased.

PEGylation with the 60K reagent had a substantial decreasing effect onthe specific activities, even at low degrees of PEGylation. Increasingthe PEGylation degree (Lys 20K br medium and Lys 20K br high conjugates)resulted in a substantial decrease in all activities, especially in theVWF:CB and VWF:FVIIIB, where only a few percent of the initialactivities could be detected for the Lys 20K br high conjugate.

PEGylation with the releasable PEG reagents resulted in a substantialdecrease in the specific activities, even at low degrees of PEGylation.The higher the MWs of the reagents were, the greater was the decrease inactivities observed. No substantial differences were found between the“short” and “long” derivatives of the same MW.

The FVIII affinity was determined assuming homogeneous 1:1 interactionbetween the immobilized rVWF and FVIII, the association and dissociationconstants were determined using the Langmuir model of the“Bioevaluation” program of the Biacore 3000 apparatus. The affinityconstant (KD) for the PEGylated rVWF-FVIII interaction remained in thesame order of magnitude as measured with non-PEGylated VWF,independently of the type or MW of the applied PEG reagent.

TABLE 3 Quantitative parameters of rVWF conjugates VWF:FVIII ProteinMeasured values binding (mg/ml) VWF:Ag VWF:RCo VWF:CB VWF:FVIIIBaffinity Samples #1 #2 (IU/ml) (IU/ml) (U/ml) (U/ml) KD (M) native rVWFn.a. 0.344 54.6 24.3 63.5 46.0 5.5E−10 133P1-1 n.d. n.d. 48.3 17.8 n.d37.8 4.8E−10 133P1-2 n.d. 0.373 49.1 22.1 n.d 47.9 2.9E−10 133P1-3 n.d.0.396 42.4 17.7 n.d 37.9 n.d. mean n.a 0.385 46.6 19.2 n.a. 41.2 3.9E−10Lys 20K br short low 0.304 0.259 21.1 11.8 19.5 13.6 7.5E−10 Lys 20K brlong low 0.450 0.529 25.3 18.7 21.7 16.8 3.9E−09 Lys 40K br short low0.228 0.234 9.1 5.5 10.4 8.8 2.3E−09 Lys 40K br long low 0.315 0.21214.3 8.1 15.8 15.8 2.6E−09 Lys 60K br short low 0.216 0.235 2.9 1.0 1.32.7 5.5E−09 Lys 60K br long low 0.410 0.459 10.3 3.5 9.9 10.9 5.4E−09control 0.163 0.145 18.4 8.0 n.d. 15.5 n.d. All results were obtainedfrom a freshly thawed sample and are the mean of at least 2measurements.

TABLE 4 Specific activities of rVWF conjugates Specific activity relatedActivity related to VWF:Ag to the rVWF protein VWF:RCo VWF:CB VWF:FVIIIBVWF:Ag VWF:RCo VWF:CB VWF:FVIIIB Samples (IU/IUAg) (U/IUAg) capacity (%)(IU/mg) (IU/mg) (U/mg) (U/mg) native rVWF 0.45 1.16 84 159 71 185 134Control #1 0.41 n.d. 88 121 50 n.d. 107 Lys 20K br short low 0.56 0.9264 69 39 64 45 Lys 20K br long low 0.74 0.86 66 56 42 48 37 Lys 40K brshort low 0.60 1.14 97 40 24 46 39 Lys 40K br long low 0.57 1.10 110 4526 50 50 Lys 60K br short low 0.34 0.45 93 13 5 6 13 Lys 60K br long low0.34 0.96 106 25 9 24 27 Control #2 0.43 n.d. 84 113 49 n.d. 95 Valueswere calculated from the measured data, shown in Table 4.

Releasable rVWF coniugates: Similar to the SDS-PAGE results of thestable conjugates, mature rVWF appeared as a prominent single band(monomer) with a MW of ˜260 kDa with some minor bands down to 150 kDa.Remaining amounts of non-PEGylated rVWF monomers were still present inthe PEG-rVWF preparations, especially in the 40K derivatives.

The PEGylation led to a band shift to higher MW which correlated withthe MW of the applied PEG reagents. No structural changes were shown forthe control preparations.

To verify the results of the protein-stained SDS-PAGE, the gels wereimmunoblotted with a polyclonal anti-human VWF antibody, as demonstratedin FIG. 9, Panel A. The unmodified native rVWF 133P1 appeared as asingle VWF monomer in the anti-VWF immunoblot with some minor,non-relevant degradation bands. No structural changes were detected inthe mock preparation (control).

All releasable conjugates showed a non-PEGylated band and some distinctbands in the higher MW range. The number of the increased bands mightrepresent mono-, di-, tri- and higher PEGylated monomers. The MW ofthese bands correlated with the MW of the applied PEG reagents.

Panel B of FIG. 9 shows the immunoblot for PEG when the blots werestained with a polyclonal anti-PEG antibody and confirmed that thehigher MW bands contained PEG. No reaction was observed for thenon-PEGylated materials rVWF 133P1 and rVWF control. Lower MW bandsappeared in all samples, most likely representing some free PEG in thesample, released during sample preparation.

Structural integrity of PEGylated rVWF shown by VWF multimer analysis:Multimer composition and the effect of PEGylation on this parameter wasinvestigated by non-reducing agarose gel electrophoresis using alow-resolution (1%) gel to determine the number of the multimers and ahigh-resolution (2.5%) gel to investigate the fine structure of themultimers.

Similar to the stable conjugates, the increase in PEG size resulted in aloss of resolution in the high MW multimer range in the low-resolutionagarose gel electrophoresis (blurred area in Panel A of FIG. 10), whichmade the exact number of the multimers difficult to determine. Theimmunoblot for PEG (FIG. 10, Panel B) demonstrated that apparently allmultimers were PEGylated, which means that each multimer contained atleast one PEGylated VWF monomer.

The high-resolution agarose gel (FIG. 11) showed a broadening and clearshift of the VWF multimers to higher molecular weights with a minor MWincrease for the Lys 20K br short low and a medium increase for the Lys20K br long low conjugates (Panel A). These data correlated with thePEGylation degree described in Table 3. Multimer analysis of the 40K andparticularly of the 60K conjugates resulted in a blurred area instead ofdistinct bands.

Panel B of FIG. 11 confirmed that apparently all multimers werePEGylated, which means that each multimer contained at least onePEGylated VWF monomer. For the 60K conjugates some lower MW bands werestained with the anti-PEG antibody, which might represent some free PEG,probably released under the electrophoretic conditions.

Determination of the FVIII-binding capacity of the PEGylated-rVWF in thepresence of unmodified rVWF under flow conditions: Hemophilia A patientsto be treated with rFVIII complexed non-covalently with the PEGylatedrVWF have normal levels of endogenous VWF, therefore the question arose,whether this VWF might compete with the PEGylated rVWF for the injectedrFVIII. To address this question, competition of FVIII complexed withPEGylated rVWF with native rVWF was measured in a Biacore system, asdescribed above with respect to determination of the FVIII-bindingcapacity of PEG-rVWF in the presence of native rVWF under flowconditions. Constant amounts of unmodified rVWF were immobilized to thesensor chip of the Biacore equipment and native or PEGylated rVWF-FVIIIcomplex, containing 5 IU/ml rFVIII, and increasing amounts of VWF:Agwere injected. The rFVIII complexed with the injected VWF was expressedas a percent of the added FVIII.

FIG. 12 shows the percent of rFVIII that remained in complex with thereleasable PEGylated rVWF conjugates as a function of the μg rVWF/IUFVIII. The star symbol (*) line shows the native rVWF, requiring thelowest VWF to FVIII ratio (approximately 1 μg/IU) to keep all FVIII in acomplex. The cross (+) line with represents an inhouse rVWF preparation(rproVWF 198) containing about 50% of proVWF and mature VWF. Thispreparation can bind less FVIII because the propeptide shades theFVIII-binding site of VWF. Bendetowicz et al. (1998) Blood92(2):529-538.

The ELISA-based VWF:FVIII-binding assay (ECA) revealed no substantialdifferences between the binding capacity of the releasable conjugates(Table 5). All showed a diminished capacity compared with the nativerVWF. All conjugates showed a 100% binding of FVIII above a ratio of 1μg VWF/U FVIII. These results correlate well with the animal models.

Susceptibility to ADAMTS13: Under physiological conditions, ultra-largemultimers of VWF are degraded by the VWF-cleaving protease (ADAMTS13),which thus plays a role in the prevention of platelet aggregation whichcould be induced by these ultra-large multimers of VWF. Because theexpressed rVWF has never been exposed to ADAMTS13, its susceptibility toADAMTS13 is an important measure of the structural integrity of an rVWFproduct. The ADAMTS13-induced physiological degradation can be simulatedin vitro by incubating rVWF and ADAMTS13 under denaturing conditions.

FIG. 13 shows the relative changes of VWF:CB activity (% of the VWF:CBactivity measured in the absence of ADAMTS13) of the conjugates as afunction of ADAMTS13 concentration. The Lys 60 K br conjugate had a verylow VWF:CB activity (Table 4). Therefore it could not be tested. Allconjugates showed a similar gradual loss of VWF:CB activity incubatedwith increasing amounts of ADAMTS13. The PEGylated conjugates showed aslightly higher susceptibility to ADAMTS13 than their parent nativerVWF. Multimer analysis demonstrated the disappearance of the highermolecular weight multimers (FIG. 14). All preparations showed similarsensitivity to ADAMTS13.

In vitro release of releasable PEG-rVWF: To investigate the kinetics ofin vitro release of the PEG moieties from rVWF, the PEGylated rVWFsamples were diluted to approximately 100 μg/ml with Hepes buffer (20 mMHepes, 150 mM NaCl, 0.5% sucrose, adjusted to pH ˜7.5 with 100 mM Tris).As a control, a native rVWF was treated the same way. All samples werekept at ambient temperature for 8 days. A sub-sample was taken every dayat the same time, aliquoted, frozen and stored at −80° C. untilanalysis.

Table 5 shows the calculated VWF:Ag/protein ratio (IU/mg). The nativerVWF 133P1 was stable over the whole time period. The VWF:Ag valuediffers at the start point (0 hours) because of the variation of theVWF:Ag to protein ratios between the different PEG-rVWF conjugates.Nevertheless all six derivatives showed an increase in VWF:Ag levelduring the incubation, albeit to different extents. With the exceptionof the Lys 60K br short low conjugate, the VWF:Ag to protein ratioreached the range of the native rVWF.

TABLE 5 Changes of VWF:Ag to protein ratio of releasable PEG-rVWF duringin vitro incubation Lys Lys Lys Lys Lys Lys Incubation 20K 20K 40K 40K60K 60K time short long short long short long rVWF (days) br low native0 92 74 75 63 51 56 98 1 119 91 77 58 59 62 103 2 84 86 70 61 66 65 80 3108 81 93 81 64 71 108 4 103 98 83 66 72 83 95 5 114 88 106 87 71 94 1126 99 113 92 87 64 n.d. 81 7 124 94 83 102 69 93 113 8 116 83 87 102 63103 97

TABLE 6 Changes in VWF:FVIIIB specific activity of releasable PEG-rVWFduring in vitro incubation Lys Lys Lys Lys Lys Lys Incubation 20K 20K40K 40K 60K 60K time short long short long short long* rVWF (days) brlow native 0 46 34 37 35 17 n.a. 81 2 60 45 40 35 32 n.a. 66 5 102 54 8263 57 n.a. 93 8 128 73 92 101 69 n.a. 84 *VWF:FVIIIB of the 60K longconjugate after release reaction in buffer could not be measured due tonon-parallel dilution curves

Table 6 shows the changes in the specific VWF:FVIIIB capacity calculatedas U/mg protein. During the incubation period a full recovery ofVWF:FVIIIB was observed for all batches evaluated.

To assess the possible degradation of rVWF and the changes in PEGylationgrade, the samples were subjected to SDS-PAGE under reducing conditionsfollowed by immunoblots with a polyclonal anti-VWF antibody and apolyclonal anti-PEG antibody (FIG. 15). As shown in FIG. 15, allPEG-rVWF derivatives showed a gradual release of PEG after in vitrorelease in buffer with a moderately increased pH (7.5-7.7).Corresponding to the release behavior of the conjugates of “short” and“long,” some differences in the PEGylation degree could be observedafter longer incubation periods. In contrast, there was no substantialincrease in the free PEG during the incubation, possibly because of thepresence of free PEG already shown in the first sample after dilutionwith the incubation buffer. No free PEG was visualized for the 20Kconjugates because the immunoblot showed only MW ranges above 75 kDa.

Pharmacokinetics of PEGylated rVWF and co-injected rFVIII in thehemophilia mouse model: FVIII-deficient knockout mice were infused witheither a mixture of rVWF/rFVIII or PEGylated rVWF/rFVIII in a targetratio of 1.6-2.1 mg rVWF to 200 IU rFVIII/kg (based on Bradford proteindetermination). In the experiments, 6 mice per time group were used foreach conjugate.

FIGS. 16-21 show the changes in plasma levels of VWF:Ag (Panel A) andFVIII activity (Panel B) after substance injection. The exact amount ofthe injected material is shown in the appropriate figure legends.

In general, all PEGylated rVWF showed improved pharmacokinetics versusthe native control (Panels A). The 60K PEG conjugates gave an increasein detectable VWF:Ag after injection, which might be an effect from therelease of PEG chains, thus making masked epitopes accessible for thedetection antibody. rFVIII, injected together with PEG-rVWF, waseliminated to a lower extent than rFVIII co-injected with native rVWF(Panels B). The degree of improvement of pharmacokinetic parameters wascalculated with statistical methods (see Tables 7 and 8).

For in vivo experiments, a control mixture (native rVWF and rFVIII) wascompared in one experimental set with one or two candidate mixtures(PEG-rVWF and rFVIII) and elimination curves were constructed (FIGS.16-21). To allow comparative analysis of releasable PEG-rVWF candidates,the elimination curves were normalized. The plasma level obtained 5minutes after application was set to 100% and all subsequent levels werecalculated relative thereto. The mean of all control groups performedthroughout the investigation (n=48 mice at each time group) is shown forcomparison in FIG. 22 together with all candidates.

The comparative analysis confirmed that all PEGylated rVWF circulatedlonger in FVIII-deficient mice, compared to native rVWF. rFVIII injectedtogether with PEG-rVWF had superior elimination characteristics over thecontrol mixture (rFVIII and native rVWF).

Area under the curve for VWF:Ag was calculated relative to the VWF:Agunits applied and also in relation to the amount of protein applied.Relative increase factors for the PEG-rVWF candidates versus control aregiven in Table 7.

TABLE 7 Increase in dose-adjusted AUC for VWF:Ag VWF:Ag AUC VWF:Ag AUCadjusted to adjusted to protein dose VWF antigen dose rVWF Sampleincrease versus control increase versus control Lys 20K br short low 3.24.3 Lys 20K br long low 2.2 3.8 Lys 40K br short low 1.9 3.9 Lys 40K brlong low 2.5 2.9 Lys 60K br short low 1.6 5.1 Lys 60K br long low 1.53.4

The area under the curve for PEG-rVWF antigen was increased for allcandidates in a range from 2.9 to 5.1 when dose-adjusted to VWF antigenunits injected. The increase was statistically significant for allcandidates. When calculated relative to the protein dose, AUC wasincreased between 1.5 and 3.2 fold, statistical significance was notcalculated.

FIG. 23 shows the dose-adjusted AUC values together with 95% confidenceintervals.

Table 8 summarizes the half-life parameters calculated for rFVIII,co-injected with PEG-rVWF candidates.

TABLE 8 Increase in pharmacokinetic parameters for co-injected rFVIIIFVIII AUC FVIII half life FVIII MRT rVWF Sample increase versus controlLys 20K br short low 2.0 s 0.9 ns 1.3 Lys 20K br long low 1.8 s 1.0 ns1.7 Lys 40K br short low 1.8 s 1.2 ns 1.5 Lys 40K br long low 1.9 s 1.3ns 1.7 Lys 60K br short low 1.6 s 1.5 ns 2.0 Lys 60K br long low 2.1 s0.9 ns 1.8 s: significant; ns: not significant

As shown in Table 8, all PEGylated rVWF candidates caused astatistically significant increase in dose-adjusted AUC for co-injectedrFVIII. FVIII half-life was not significantly changed by all candidates.Mean residence time was elevated by a factor between 1.2 and 2.0,however significance could not be calculated with the statistical modelused.

FIG. 24 shows the dose adjusted AUC values and the terminal half lifefor FVIII together with the 95% confidence intervals.

Dose adjusted AUC for co-injected FVIII was higher with all PEGylatedrVWF candidates, compared to the native rVWF control. In contrast, FVIIIterminal half life in the presence of PEGylated rVWF was very similar tohalf life obtained with rFVIII and native rVWF control, which reflectsthe parallel run of the FVIII activity curves at later time points inFIG. 22.

Mean residence time for FVIII (FIG. 31) was always increased whenco-injected with PEGylated rVWF candidates

In summary, all PEGylated rVWF conjugates preserved the multimericstructure without any degradation. In contrast all functional activitieswere decreased. The low VWF:RCo and VWF:CB activity has no effect on thechaperon function of VWF. It might even have the advantage of avoidingplatelet adhesion. The diminished VWF:FVIIIB capacities measured in astatic assay was improved under shear conditions, suggesting thatPEG-rVWF is capable of carrying an appropriate amount of FVIII in thecirculation. In conclusion, taking all in vivo data together, allPEGylated rVWF candidates show an improved pharmacokinetic profile forVWF:Ag in FVIII-deficient mice, which is paralleled by an improvement ofpharmacokinetic profile for co-injected rFVIII.

EXAMPLE 6 In Vitro and In Vivo Experiments of PEGylated FVIII

The native recombinant FVIII, was an Advate rAHF-PFM [AntihemophilicFactor (Recombinant) Plasma/Albumin Free Method bulk drug substance], alicensed lyophilized drug product of Baxter AG. This rFVIII bulksubstance was formulated in a buffer of 50 mM HEPES, 5 mM CaCl₂, 350 mMNaCl and 0.1% Polysorbate 80 adjusted to pH 6.9. The analytical data ofthis rFVIII are given in Table 9. Protein content was determined usingthe BCA assay (Pierce, Rockford, Ill., USA) and specific activity isexpressed as the ratio of FVIII chromogenic activity (IU)/protein (mg).The rFVIII bulk contained less than 2.3 μg VWF:Ag/1000 IU of rFVIII.SDS-PAGE performed under reducing conditions followed by immunoblot witha polyclonal antihuman FVIII antibody showed the intact domain structureof FVIII (FIG. 26).

TABLE 9 Analytical data of the native rFVIII MOQ HEPES 01-E NativerFVIII MOQ_HEPES_01-E Protein BCA (mg/ml) 3.020 FVIII: Chrom activity(IU/ml) 19167 Specific activity (IU/mg) 6347 FVIII:Ag (IU/ml) 19698VWF:Ag (IU/ml) 4.5

PEG-rFVIII conjugates with releasable bonds via the amino groups of thelysine residues of rFVIII were prepared in accordance with Examples 2A,2A1, 2B, 2B1, 2C, 4A, 4B, 4B1, and 4C. The branched PEG derivatives hadmolecular weights of 20K, 40K and 60K, each of them with two differentrelease characteristics (short and long release time). To investigatethe possible effect of the conjugation conditions with the releasablePEG reagents, a mock preparation (control) was also manufactured whichran through the whole process but was not PEGylated.

TABLE 10 PEGylation degree of the PEG-rFVIII preparations PEG/FVIII*PEG/FVIII** Protein colorimetric HPLC Material Name (mg/ml) (mol/mol)(mol/mol) Free PEG Lys 20K br short 0.290 12.7 12.8 <0.01% Lys 20K brlong 0.265 5.9 11.1 not detectable resynthesized 0.320 — 8 <0.01% Lys40K br short 0.400 107 — <0.01% resynthesized 0.100 — 11 <0.01% Lys 40Kbr long 0.120 14.6 — <0.01% resynthesized 0.220 — 10.1 <0.01% Lys 60K brshort 0.500 9.8 — <0.01% Lys 60K br long 0.120 12.3 11.3 <0.01% Theprotein content of the PEG-rFVIII was determined using the “DC Proteinassay” of Bio-Rad (Hercules, CA, USA) with the unmodified rFVIII asstandard. *The number of PEGs per molecule was determined using acolorimetric determination **The number of PEGs per molecule wasdetermined by an HPLC method —: no data avilbale The remaining free PEGwas determined by barium-iodide staining of the non-reduced SDS-PAGE.

Due to the extended in vitro and in vivo analytical testing, threeconjugates had to be resynthesized to complete the investigations. Somedifferences in the PEG to FVIII ratio were observed between theresynthesized conjugates and the first batches for the 20K br long andLys 40K br long PEG-rFVIII derivatives, which might be explained by thedifferent analytical methods used. As investigated by barium-iodinestaining, no free PEG remained in any sample solution.

Determination of FVIII activity: FVIII activity was determined with achromogenic method. In the assay, the FVIII-containing sample was mixedwith thrombin, activated factor IX (FIXa), phospholipids and factor X(FX), in a buffer containing calcium. FVIII is activated by thrombin andsubsequently forms a complex with phospholipids, FIXa and calcium ions.This complex activates FX to FXa, which in turn cleaves a specificchromogenic substrate releasing para-nitroaniline (pNA) resulting in acolor reaction.

For analysis of PEG-rFVIII conjugates, samples and the reference werepre-diluted to approximately 1 IU/ml FVIII:Chrom activity in a humanFVIII-deficient plasma and further diluted with the dilution buffer to arange from 0.5 to 0.008 IU/ml FVIII:Chrom. The time course of the pNAreleased from the substrate was measured with a microplate reader at 405nm using the kinetic mode. The slope of the reaction is proportional tothe FVIII concentration in the sample. The FVIII concentration in thesamples was calculated relative to a recombinant FVIII concentratestandard, calibrated against the World Health Organization (WHO)concentrate reference (WHO 6) and expressed in IU/ml. The quantificationlimit of the assay was 0.03 IU/ml of FVIII.

Determination of FVIII antigen by enzyme-linked immunosorbent assay(ELISA): The FVIII antigen level was determined according to themanufacturer's instructions with some minor modifications using theassay kit obtained from Cedarlane (Cedarlane Laboratories, Hornby,Ontario, Canada). High-binding 96-well ELISA plates (Costar 3590,Corning Incorporated, NY, USA) were coated with 100 μl/well of apolyclonal anti-human FVIII antibody and incubated for two hours at roomtemperature. Samples were diluted from 0.0078 to 0.5 IU/ml FVIII:Ag withthe dilution buffer from the kit. Plates were then washed withphosphate-buffered saline (PBS; 6.5 mM disodium hydrogenphosphatedihydrate, 1.5 mM potassium dihydrogen phosphate, 140 mM NaCl, pH 7.2)containing 0.05% Tween-20 (PBST). 100 μl of the diluted samples wereadded to the plates and incubated for two hours at room temperature.After a washing step with PBST, 100 μl/well of peroxidase conjugatedpolyclonal anti-human FVIII antibody (#EIA8-0015R1, CedarlaneLaboratories, Hornby, Ontario, Canada) were added to the plates.Peroxidase activity was detected by using tetramethyl-benzidine (TMB) assubstrate (Bio-Rad, Hercules, Calif., USA). The developed colorintensity was measured with an ELISA reader at 450 nm. As a standard, ahuman normal plasma pool (coagulation reference, lot IR920031, Baxter)and as control a recombinant rFVIII bulk (Advate #B0206000-05/01) wasused. The FVIII:Ag concentration was calculated relative to the standardpreparation and expressed as FVIII:Ag IU/ml.

Measurement of VWF-FVIII affinity by surface plasmon resonancetechnology: Native rVWF was immobilized on the flow cells of a CM5sensor chip of a Biacore 3000 (Biacore AG, Uppsala, Sweden) apparatus toa constant level according to the instructions of the manufacturer. Aseries of dilutions of native and PEG-rFVIII samples were then appliedto the chip using the “kinject” mode, allowing three minutes for theassociation and ten minutes for the dissociation of FVIII. After each ofthese cycles FVIII was removed from the chip (“regeneration”) and theexperiment was repeated with a new FVIII sample.

SDS-PAGE and immunoblot for FVIII: FVIII samples (100 mIU equal to 10 ngprotein per lane) were applied to gradient (4-12%) Bis-Tris gels andelectrophoresis was done under mild reducing conditions, followed bystandard blotting procedures onto a polyvinylidene difluoride (PVDF)membrane. To visualize the FVIII bands, a polyclonal anti-human FVIIIantibody (CL20035A; Cedarlane Laboratories, Hornby, Ontario, Canada), amonoclonal anti-human heavy chain-A2 domain antibody (OBT0037, OxfordBiotechnology, Oxford, U.K.) or a monoclonal anti-human light chain-A3domain antibody (10104; QED Bioscience Inc, San Diego, Calif., USA) wasused as the primary antibody. As a secondary antibody, an alkalinephosphatase (ALP)-labeled rabbit anti-sheep IgG (H+L) (A130-101AP,Bethyl Laboratories, Inc, Montgomery, Tex., USA) was applied for thepolyclonal antibody and an alkaline phosphatase (ALP)-labeled goatanti-mouse IgG (H+L) (A90-216AP, Bethyl Laboratories, Inc, Montgomery,Tex., USA.) for the monoclonal antibodies. The blots were developed withthe ALP color development kit of Bio-Rad (Hercules, Calif. USA). A fullrange rainbow marker (250-10 kDa, GE-Healthcare, Little Chalfont,Buckinghamshire, UK) was used as the molecular weight standard.

SDS-PAGE and immunoblot for PEG: FVIII samples (300 mIU equal to 30 ngprotein per lane) were applied to gradient (4-12%) Bis-Tris gels andelectrophoresis was done under reducing conditions, followed by standardblotting procedures onto a PVDF membrane. To visualize the PEG, apolyclonal rabbit anti-human PEG antibody was used as the primaryantibody. The anti-PEG antibody was raised in rabbits by immunizationwith a PEGylated protein. The IgG fraction of the rabbit serum waspurified by affinity chromatography on Protein G Sepharose 4B(GE-Healthcare, Uppsala, Sweden) followed by specific negativeimmunoabsorption. An alkaline phosphatase (ALP)-labeled goat anti-rabbitIgG (A120-201AP, Bethyl Laboratories Inc., Montgomery, Tex., USA) wasapplied as a secondary antibody. The blots were developed with the ALPcolor development kit of Bio-Rad (Hercules, Calif. USA). A full rangerainbow marker (250-10 kDa, GE-Healthcare, Little Chalfont,Buckinghamshire, UK) was used as molecular weight standard.

FIXa-cofactor activity assay: Untreated or thrombin-activated nativerFVIIIa and PEG-rFVIII samples diluted to 1 IU/ml (according to theirchromogenic activities) in the presence of a thrombin-specific inhibitor(Pefabloc TH, Penthapharm, Basel, Switzerland) were added to a preparedmixture of FIXa, FX, phospholipid (PL)-vesicles [composed of 60%phosphatidylcholine (PC) and 40% phosphatidylserine (PS), both fromAvanti Polar Lipids Inc (Alabasta, Ala., USA)] and CaCl₂. This reactionmix was incubated at 37° C. to allow complex formation and subsequentFXa generation. Subsamples were withdrawn at defined intervals up to 30minutes and added to a chromogenic substrate, which is selectivelycleaved by FXa. The substrate buffer containedethylenediaminetetraacetic acid (EDTA) to stop any further FXageneration. After 15 minutes of incubation, the reaction was terminatedby the addition of acetic acid. The absorbance at 405 nm (A405) which isproportional to the FXa concentrations, was measured in an ELISA reader.A reference curve was constructed by using a purified FXa (HFXa 1011,Enzyme Research Laboratories, Swansea, UK) and the absorbance valueswere converted to FXa concentration.

Thrombin-activated rFVIII (rFVIIIa) was prepared freshly for each testby incubating 1 IU/ml native or PEG-rFVIII with 1 nM thrombin for oneminute at 37° C. and the reaction was stopped by adding 10 μM of athrombin-specific inhibitor (Pefabloc TH, Penthapharm, Basel,Switzerland).

The time course of FX activation (FIG. 27) was drawn and analysed asfollows: The maximum rate of FX activation was calculated by determiningthe slope of the linear part of the curve and was expressed as nMFXa/min. The lag phase was determined by calculating the X-axisintercept of the linear part of the curve. The maximum activity wasdetermined as the mean FXa concentration measured between 20 and 30minutes and the half maximum time (t_(1/2)) was calculated using thefollowing formula: t_(1/2)=(maximum AFXa/2+lag phase time*slope)/slope.All parameters were calculated by using the internal functions ofMicrosoft Excel.

Kinetics of thrombin-mediated activation and inactivation of FVIIImeasured by the FIXa cofactor activity assay: Native rFVIII andPEG-rFVIII were diluted to 1 IU/ml FVIII activity (according to theirchromogenic activities) with 25 nM HEPES; 175 mM NaCl, 5 mg/ml bovineserum albumin (BSA) pH 7.35 buffer and incubated with 0.5 nM thrombin at37° C. Subsamples taken at various time points up to 40 minutes wereadded to aliquots of prepared mixtures of FIXa, FX, phospholipid(PL)-vesicles (composed of 60% PC and 40% PS), CaCl₂ and a thrombininhibitor to stop further activation of FVIII. These reaction mixes wereincubated for three minutes at 37° C. to allow FXa to generate. Asubsample of this mixture was added to a chromogenic substrate, which isselectively cleaved by FXa. The FXa concentration was determined asdescribed above with respect to FIXa-cofactor activity assay and plottedagainst the incubation time of FVIII with thrombin. The rate ofinactivation had been quantitatively evaluated from the ascending partof the curves by fitting them with a single exponential using theinternal functions of Microsoft Excel.

Thrombin Generation Assay (TGA): A severe hemophilia A plasma (FVIIIactivity <1%) obtained from George King Bio-Medical (Overland Parks,Kans., USA) was spiked with 0.0025 to 1 μg/ml native or PEG-rFVIII andthrombin generation was measured with the Technothrombin TGA kit(Technoclone, Vienna, Austria) as described by the manufacturer. Thereaction was triggered by a relipidated tissue factor (TF) preparation(TFPL RB reagent) containing low TF and low PL concentrations. An amountof 10 μl of this TFPL solution was pipetted to 40 μl FVIII-deficientplasma, without FVIII or supplemented with FVIII and 50 μl of TGAfluorescence substrate, into the wells of an ELISA plate. The plate wasplaced into a Microplate Fluorescence Reader FL800 (Bio-TEK Instruments,Winooski, Vt., USA). The increase in the fluorescence intensity, whichis proportional to the concentration of the generated thrombin, wasmonitored continuously at 37° C. by automatic reading every minute up to120 minutes using an excitation wavelength of 360 nm and an emissionwavelength of 460 nm.

Because thrombin substrate was present in the assay mixture, curves wereseen that represent the accumulated effect of all the thrombin that wasgenerated and split the fluorogenic substrate during the reaction.Therefore, the rate of increase in the fluorescence intensity (the firstderivative of the curve), which reflects the actual effective thrombinconcentration, was calculated for each reading (FU/min) and converted tothrombin-equivalent concentrations (nM) using a reference curve preparedby measuring the rate of substrate conversion by a purified humanthrombin. The thrombin generation curves were drawn as the thrombinconcentration versus time, and the quantitative parameters (peakthrombin, onset time, and peak time) were calculated by the built-in KC4software (Bio-TEK Instruments, Winooski, Vt., USA) of the reader.

APC-mediated FVIII and FVIIIa inactivation: Untreated orthrombin-activated native and PEG-rFVIII samples were diluted to 1 IU/ml(according to their chromogenic activities) and incubated with 0.05 U/mlactivated protein C (APC) in the presence of 10 μM PL vesicles (composedof 60% PC and 40% PS; both from Avanti Polar Lipids Inc, Alabasta, Ala.,USA) and 5 mM CaCl₂. In the control experiments the rFVIII samples wereincubated in the absence of APC. Sub-samples were taken at defined timepoints to determine the residual active FVIII or FVIIIa by measuring itsFIXa cofactor activity as described above with respect to FIX-cofactoractivity assay.

The thrombin-activated FVIII (FVIIIa) was prepared freshly for each testby incubation of the 2 IU/ml native or PEG-rFVIIII samples with 1 nMthrombin (#2311PL, Enzyme Research Laboratories, Swansea, UK) for 1minute at 37° C. The reaction was stopped by adding 10 μM of athrombin-specific inhibitor (Pefabloc TH, Penthapharm, Basel,Switzerland).

Mouse model: As a hemophilia model, FVIII-knockout mice were used. Themice suffer from severe hemophilia A (FVIII<0.01 IU/ml) but have normallevels of VWF (approximately 0.15 IU/ml relative to human VWFreference), mimicking human hemophilia A.

Application of FVIII: The same recombinant FVIII bulk used forconjugation was used as a control substance (rFVIII MOQ_HEPES_(—)01E).The bulk was stored in aliquots frozen below −60° C. and thawed beforeuse. PEG-rFVIII candidates or the native rFVIII control were thawed andmixed with 20 mM HEPES, 150 mM NaCl, 3.2% mannitol, 0.8% trehalose, 2.5mM CaCl₂, 1% human albumin, pH 7.4 buffer to achieve appropriateconcentrations for infusion. The FVIII solutions were aliquoted, frozenat −20° C. and thawed just before the application. The target dose was200 IU/kg FVIII:Chrom. The concentrations were measured again from thethawed samples and the applied doses were calculated. Doses are given inthe figure legends in the results section. Seven to ten ml/kg bodyweightwere injected via the tail vein and groups of 6 mice were bled by heartpuncture after six minutes, 3, 6, 9, 16 and 24 hours, and if necessary,after 32 hours. Nine volumes of blood were mixed with 1 volume of 3.8%sodium citrate, and immediately centrifuged at 3000 g for ten minutes.The supernatant was again centrifuged at 3000 g for five minutes, plasmawas separated, frozen in aliquots and stored below −60° C. for analysis.

Determination of FVIII activity in mouse plasma: FVIII activity wasdetermined following the assay principle described above. The timecourse of the pNA released from the substrate was measured with amicroplate reader at 405 nm using the kinetic mode. The slope of thereaction is proportional to the FVIII concentration in the sample. TheFVIII concentration in the samples was calculated relative to a humanplasma reference preparation, calibrated against the WHO plasmareference (5^(th) IS for FVIII and VWF in human plasma, NIBSC #02/150)and expressed in IU/ml. The quantification limit of the assay was 0.03IU/ml of FVIII.

Calculation of the circulating half-life parameters of human VWF andFVIII: For analyzing FVIII levels, the concentrations for t₀=0 hours wasset to zero as FVIII-deficient mice were studied. FVIII levels over timewere summarized using pharmacokinetic parameters AUC from 0 to 24 hours,terminal elimination rate and mean residence time.

Area under the concentration vs. time curve (AUC) from 0 to 24 hours:The area under the concentration vs. time curve (AUC) from 0 to 24 hourswas calculated by the linear trapezoidal rule using the arithmetic meansof the concentrations observed at individual time points. A linearrelation was assumed to exist between dose and AUC. On this assumption,the AUCs for different items were adjusted for different dosesadministered. Dose adjustment was performed by dividing the calculatedAUC by the dose per kg body mass administered.

Terminal elimination rate: The terminal elimination rate (λ) wasestimated using the arithmetic mean of the natural logarithms ofindividual concentrations at the last three time points modified with abias correction as suggested in Wolfsegger. See Wolfsegger et al. (2005)J. Pharmacokinet. Pharmacodyn. 32(5-6):757-766.

Mean residence time: Mean residence time (MRT) was calculated asAUMC_(0-infinity) divided by AUC_(0-infinity). AUMC_(0-infinity) andAUC_(0-infinity) were calculated by the linear trapezoidal rule usingthe arithmetic means of the concentrations observed for different timepoints plus a three-point tail area correction. The tail area correctionwas calculated by log-linear fitting on the arithmetic means observed atthe last three time points per item.

Functional parameters of PEGylated rFVIII: The potency of modified FVIIIwas measured by its chromogenic activity. Under the assay conditions,FVIII is activated by thrombin and thus the assay reflects its maximumpotency to enhance the FIXa-mediated FX activation. To distinguishbetween biological active and inactive FVIII, the FVIII:Ag wasdetermined by ELISA, as described in the experimental section. Tocompare the specific activities of the different conjugates bothparameters were related to the protein content of the products. Table 11summarizes the measured values.

TABLE 11 Quantitative parameters of PEGylated rFVIII conjugatesVWF:FVIII Measured values binding Protein FVIII:Ag FVIII:chrom affinitySamples (mg/ml) (IU/ml) (IU/ml) KD (M) native rFVIII 3.02 19698 191671.5E−09 FVIII control 0.170 1120 820 5.5E−10 Lys 20K br short 0.290 82277 2.4E−10 Lys 20K br long 0.265 116 231 2.9E−10 0.320 114 281 n.d. Lys40K br short 0.400 122 512 3.4E−10 0.100 33 111 n.d. Lys 40K br long0.120 25 130 1.2E−10 0.220 32 183 n.d. Lys 60K br short 0.500 142 5261.2E−09 Lys 60K br long 0.120 65 272 2.1E−10 (All results were obtainedfrom a freshly thawed aliquot and are the mean of at least 2measurements.)

The FVIII affinity for VWF was determined using the Biacore 3000 systemas described above with respect to measurement of VWF-FVIII affinity bysurface plasmon resonance technology with an immobilized native rVWF(rVWF 133P1) and the sample (native rFVIII or PEG-rFVIII conjugates) inthe fluid phase. Assuming a homogenous 1:1 interaction between VWF andFVIII, the association and dissociation constants were determined usingthe Langmuir model of the “Bioevaluation” program of Biacore. Norelevant differences for the affinity constant (KD) with VWF between thenative and the PEG-rFVIII were found. Only an approximate evaluationcould be performed because the PEG-rFVIII conjugates did not give anoptimal fitting, either with this or any other interaction-models,possibly because of some conformational changes of rFVIII due to thePEGylation. The ratios of the measured values and the specificactivities are summarized in Table 12.

TABLE 12 Specific activities of PEG-rFVIII conjugates Ratio SpecificActivity FVIII:Chrom related to the rFVIII protein to FVIII:AgFVIII:Chrom FVIII:Ag Samples (IU/IU) (IU/mg) (IU/mg) native rFVIII 0.976347 6523 FVIII control 0.73 4824 6588 Lys 20K br short 3.38 955 283 Lys20K br long 1.99 872 438 Resynthesized 2.46 878 356 Lys 40K br short4.20 1280 305 Resynthesized 3.36 1110 330 Lys 40K br long 5.20 1083 208Resynthesized 5.71 832 145 Lys 60K br short 3.70 1052 284 Lys 60K brlong 4.18 2267 542 Values were calculated from the measured data, shownin Table 3.

All PEG-rFVIII conjugates had a markedly reduced FVIII activity comparedwith the native rFVIII. However, the FVIII specific activity was alsoslightly reduced for the FVIII control, which was not PEGylated but ranthrough the whole process.

The decrease in activity was not related either to the MW or to thecharacteristics of the PEG reagents. For example, the Lys 60K br longconjugate had an approximately 65% reduced FVIII specific activity,while the specific activity of the Lys 20K br long conjugates wasreduced to approximately 14% compared with the native rFVIII. Thecorrelation between PEGylation degree and specific activities cannot beassessed, because two different methods for determination of the degreeof PEGylation were used throughout the analytical characterization,except for the Lys 20K br long conjugate, where data obtained with theHPLC-method are available for both the original conjugate and theresynthesized material. Although the resythesized Lys 20K br long had alower PEGylation degree than the original conjugate, both had similarspecific activities.

The FVIII antigen to protein ratio was below 10% in all conjugates.Because two polyclonal antibodies were applied in the assay describedabove with respect to determination of FVIII antigen by enzyme-linkedimmunosorbent assay (ELISA), this decrease indicates a strong shadingeffect of the PEG moieties throughout the molecule. The FVIIIchromogenic activity (FVIII:Chrom) to FVIII:Ag ratio was elevated, whichmight suggest that despite of the strong coverage of some epitopesPEG-rFVIII can be activated or a partial release of the PEG moietiesoccurred immediately under the activity determination conditions,possibly due to the effect of thrombin in the reagents.

The detailed biochemical characterization was carried out on the firstbatches of each PEGrFVIII conjugate. Due to the lack of the originalmaterial, the resynthesized conjugates were used for the investigationof APC-mediated inactivation of rFVIII (described below under“APC-mediated inactivation of FVIII and FVIIIa”) and for theinvestigation of the in vitro hydrolysis in a human FVIII-deficientplasma (described below under “in vitro release of releasable PEG-rFVIIIin a human FVIII-deficient plasma”).

The domain structure of the PEG-rFVIII conjugates was visualized bynon-reducing SDS-PAGE followed by immunoblot with a polyclonalanti-human FVIII antibody (FIG. 28, Panel A). For the assessment ofsuccessful PEGylation, the gels were immunoblotted with a polyclonalanti-PEG antibody, as demonstrated in FIG. 28, Panel B. All samples wereapplied to the gel according to the measured protein value.

The characteristic domain structure of FVIII was not affected byPEGylation in any of the PEGrFVIII conjugates. As a result ofPEGylation, new, high MW bands appeared with a concomitant decrease inthe intensity of some heavy chain (HC)-B domain bands. Staining with theanti-PEG antibody confirmed successful PEGylation. No degradationproducts were seen on the gels.

Because the polyclonal antibody did not have the same affinity for thedifferent domains, the gels were also immunoblotted with antibodiesspecific against the HC-A2 fragment and the light chain(LC)-A3 domain(FIG. 29).

FIG. 29 Panel A shows the PEG MW-dependent increase of the HC-containingbands. The antibody also shows some 90-kDa intact HC bands, without anyfurther degradation bands. The rFVIII used as the starting materialcontained an “extended” light chain, which was also detected by theanti-LC antibody (FIG. 29, Panel B). A MW increase is observed for boththe 80-kDa and the 160-kDa bands in the immunoblot, suggesting that thelight chain has also been PEGylated, albeit possibly to a lower extent.Some lower MW degradation products appeared in the 40K PEG-rFVIIIconjugates.

Effect of PEGylation of rFVIII on FIXa-cofactor activity: TheFXa-generation assay, also known as the FIXa-cofactor assay, is based onthe fact that both FVIII and thrombin-activated FVIII (FVIIIa) form acomplex with FIXa on an appropriate phospholipid (PL) surface in thepresence of Ca⁺⁺ ions, which rapidly activates FX. See Elodi et al.(1981) Thrombosis Research 21:695. The kinetics of the assembly andactivity of the complex is regulated by FVIII and is a sensitive measureof the functional integrity of the FVIII molecule. The PEG-rFVIIIconjugates as well as the FVIII control and a native rFVIII were dilutedto 1 IU/ml according to the measured FVIII chromogenic activities andadded to the prepared mixture of FIXa, FX, PL-vesicles and calciumchloride. At defined intervals up to 30 minutes subsamples werewithdrawn and the generated FXa determined as described above withrespect to FIXa-cofactor activity assay. However, even if all rFVIIIconjugates were diluted to 1 IU/ml, there were slight differences in themaximum FXa achieved (Table 13). Therefore, for a better visualcomparison of the time course of FX activation, FXa activity wasexpressed as a percent of the maximum FXa activity (FIG. 30).

Without thrombin activation, all PEG-rFVIII conjugates showed a delayedcomplex formation and a slower rate of FX activation than that of bothnative rFVIII and the FVIII control. No relevant differences in the rateof FX activation were observed between the conjugates, except that theLys 40K br long showed a slightly more reduced FX activation rate. Thecontrol seemed to be slightly more active than the native rFVIII.

TABLE 13 Quantitative parameters of the FIXa-cofactor activity withoutthrombin activation without thrombin activation Lys Lys Lys Lys Lys Lys20K 20K 40K 40K 60K 60K Native rFVIII br br br br br br rFVIII controlshort long short long short long lag phase (min) 2.4 1.8 4.2 4.0 3.9 4.33.8 3.7 maximum rate (nM FXa/min) 6.7 8.8 2.6 2.1 2.0 1.3 2.0 2.3t_(1/2) of maximum (min) 4.6 4.0 10.8 10.8 11.4 12.3 11.3 9.8 maximumFXa (nM) 30.1 39.6 33.6 28.7 30.6 21.6 30.2 28.8

Thrombin-activated rFVIII (rFVIIIa) was prepared from all products byincubating 1 IU/ml native or PEG-rFVIII with 1 nM thrombin for oneminute at 37° C. The reaction was stopped by adding 10 μM of athrombin-specific inhibitor (Pefabloc TH, Penthapharm, Basel,Switzerland) and the cofactor activity of rFVIIIa was measured as forthe non-activated rFVIII.

For showing the time course of FX activation obtained with the differentPEG-rFVIII conjugates, FXa activity was expressed as a percent of themaximum FXa activity (FIG. 31). The quantitative kinetic parameters aresummarized in Table 14.

TABLE 14 Quantitative parameters of the FIXa-cofactor activity afterthrombin activation after thrombin activation Lys Lys Lys Lys Lys Lys20K 20K 40K 40K 60K 60K Native rFVIII br br br br br br rFVIII controlshort long short long short long lag phase (min) 1.15 1.13 0.98 1.101.55 1.36 1.37 1.16 maximum rate (nM FXa/min) 4.24 9.79 6.22 5.07 5.085.18 4.94 5.09 t_(1/2) of maximum (min) 4.24 2.85 4.06 4.60 5.13 4.614.94 4.40 maximum FXa (nM) 25.1 33.7 38.3 35.5 36.3 33.6 35.3 32.9

After activation with thrombin, all PEG-rFVIII conjugates showed a rateof FX activation similar to that of the native rFVIII or the control,which still had an enhanced activity. Also a slight increase had beenobserved in the maximum FXa-generating capacity.

Kinetics of thrombin-mediated activation and inactivation of FVIIImeasured by the FIXa cofactor activity assay: The time course ofthrombin activation and inactivation was measured with the FIXa-cofactoractivity assay. Samples containing 1 IU/ml native or PEG-rFVIII wereincubated with 0.5 nM thrombin. Subsamples were withdrawn before theaddition of thrombin and at intervals afterwards up to 40 minutes andadded to a prepared mixture of FIXa, FX, PL-vesicles, calcium chlorideand a thrombin inhibitor to stop further reaction on FVIII. Thisreaction mix was incubated for three minutes because at this time pointthere was only a minimum FXa formation without thrombin and about 40% ofmaximum activity was already reached after full activation by thrombin.

FIG. 32 shows the time course of thrombin activation and inactivationfor the native and PEG-rFVIII conjugates. Table 15 shows the relativefirst order inactivation rates compared with that of the native rFVIII.

TABLE 15 Rate Constants of thrombin activation Sample Relative k′ FVIIIcontrol 0.72 Lys 20K br short 0.35 Lys 20K br long 0.39 Lys 40K br short0.48 Lys 40K br long 0.44 Lys 60K br short 0.46 Lys 60K br long 0.51

In the presence of 0.5 nM thrombin, both the native rFVIII and thecontrol showed a rapid activation with a maximum activity within 2minutes followed by a fast inactivation that was almost completed within20 and 30 minutes, respectively. In accordance with the FXa generationcharacteristics, the control could be more activated by thrombin andshowed a slightly slower inactivation rate. The PEG-rFVIII candidatesshowed a slightly slower activation rate reaching a maximum between 3 to5 minutes and a substantially decreased inactivation rate with residualFIXa-cofactor activities.

APC-mediated inactivation of FVIII and FVIIIa: Native and PEGylatedFVIII (1 IU/ml diluted according to FVIII:Chrom activity) were incubatedwith 0.05 U/ml activated protein C (APC) either with or withoutpre-activation by thrombin in the presence of PL-vesicles and CaCl₂ (asdescribed above). The residual FVIII activity after APC inactivation wasdetermined by measuring the FIXa cofactor activity, similar as describedfor investigating the thrombin-mediated activation and inactivationkinetics. Subsamples of the APC-rFVIII mixes were withdrawn at intervalsup to ten minutes and added to a prepared mixture of FIXa, FX,PL-vesicles, and calcium chloride. The mixtures were incubated for tenminutes when non-activated and for five minutes, when thrombin-activatedPEG-rFVIII was investigated. These incubation times were based on thetime courses of FXa generation measured in the presence of non-activatedand activated PEG-rFVIII conjugates (FIGS. 30 and 31); at the chosentime points about 70% of the full activity has been already reached withthe PEG-conjugates. In the appropriate control experiments native andPEG-rFVIII were incubated in the absence of APC. FIGS. 33 and 34 showthe time course of inactivation, where FXa activity was expressed as apercent of the FXa measured during the first minute in the appropriatecontrol mixtures incubated without APC. Table 16 and 17 show thecalculated relative first order rate constants compared with thatmeasured for the native rFVIII.

TABLE 16 Rate Constants of APC-mediated inactivation Sample Relative k′Lys 20K br short 0.46 Lys 20K br long 0.56 Lys 40K br short 0.44 Lys 40Kbr long 0.35 Lys 60K br short 0.52 Lys 60K br long 0.38

When non-activated native rFVIII was incubated with 0.05 U/ml APC, afirst order rate inactivation was observed, with a k′ of 0.220*min⁻¹. Incontrast, the PEG-rFVIII conjugates first showed a transient increase intheir FIXa-cofactor activities followed by a first order inactivation,albeit at an approximately 50% slower rate. There were no such changesin the samples incubated in the absence of APC. Both native andPEG-rFVIII conjugates remained stable (insert in FIG. 33). No data areavailable for the control due to the lack of test material at the testtime point.

TABLE 17 Inactivation Rate Constants Sample Relative k′ Lys 20K br short0.50 Lys 20K br long 0.50 Lys 40K br short 0.55 Lys 40K br long 0.54 Lys60K br short 0.54 Lys 60K br long 0.64

Incubation of thrombin-activated native or PEG-rFVIII showed first orderinactivations (FIG. 34) with approximately 50% slower rates for thePEG-rFVIII conjugates (Table 9). Thrombin-activated native rFVIIIa andPEG-rFVIIIa conjugates remained stable or showed only a negligibledecrease in FIXa-cofactor activity in the absence of APC.

Effect of PEGylation on the thrombin-generating capacity of FVIII inFVIII-deficient plasma: A plasma sample of a severe hemophilia A patientwith FVIII activity below 0.01 U/ml (<1%) was spiked in vitro withincreasing amounts of native and PEG-rFVIII in the range of 0.0025 and0.1 μg/ml, corresponding to an activity range of the intact FVIII of0.025 to 1 IU/ml. Thrombin generation triggered with low concentrationsof TF and PL complex was measured as described in the experimentalprocedures. As shown in FIG. 35 (Panel A) the addition of native rFVIIIdose-dependently improved the impaired thrombin generation ofFVIII-deficient plasma. The improvement resulted in a shortening of theonset time and peak time and an increase in the peak thrombin, whichshowed a linear dose-response with the logarithmic of FVIIIconcentrations, as drawn in Panel B of FIG. 35. This correlation impliesthat the most effect occurs in the low concentration range.

FIG. 36 shows the thrombin generation curves (Panels A-F) obtained withthe PEG-rFVIII samples in the FVIII-deficient plasma and thedose-response curves (Panel G) of the peak thrombin values.

All PEG-rFVIII conjugates corrected the impaired thrombin generation ofFVIII-deficient plasma in a dose-dependent manner, however with minimaleffect below 0.01 pg/ml plasma. Above this concentration paralleldose-response curves with the native rFVIII were measured, whichindicates that more FVIII is needed to achieve the same peak level asthe native rFVIII. The Lys 60K br long conjugate seemed to have a higheractivity in this assay, especially in the higher concentration range.

In vitro release of releasable PEG-rFVIII at increased pH: Toinvestigate the kinetics of in vitro reelase of the PEG moieties fromrFVIII, the PEGylated rFVIII samples were incubated in the originalrFVIII buffer (50 mM HEPES, 5 mM CaCl2, 0.1% Polysorbate 80, 350 mMNaCl, pH ˜6.9) adjusted to pH 8.1 with a 1/10 volume of 0.1 M NaOH. As acontrol, a native rFVIII and the un-PEG-rFVIII control was treated thesame way. All samples were kept at ambient temperature. A sub-sample wastaken at different time points and changes in FVIII:Ag, FVIIIchromogenic activity were measured. The structural changes wereinvestigated by SDS-PAGE followed by immunoblotting with FVIII andPEG-specific antibodies.

Changes in FVIII:AG and chromogenic activity during in vitro release ofthe PEG moiety: FIG. 37 shows the changes in FVIII-specific activity andFIG. 38 in FVIII:Ag, both expressed as IU/mg protein.

The native rFVIII and the shipping control showed a continuous decreaseof activity and antigen levels. In contrast, both the activity andantigen levels of the PEGylated rFVIII conjugates gradually increased inthe first 48 hours and after a plateau decreased again. The highestrelative activity increase (2.4 fold) was achieved for the Lys 40K brshort conjugates. The fastest increase with the shortest plateau wasobserved for the two 60K conjugates.

Structural changes in PEG-rFVIII during in vitro release of the PEGmoiety: To visualize structural changes upon incubation at higher pH ofpH 8.1, the samples were subjected to SDS-PAGE under reducing conditionsfollowed by immunoblots with polyclonal anti-human FVIII antibody (FIG.39), monoclonal anti-human heavy chain A2 domain antibody (FIG. 40) andpolyclonal anti-PEG antibody (FIG. 41). Native rFVIII and the FVIIIcontrol showed a continuous decrease in FVIII activity and FVIII:Aglevel, which corresponds to the degradation of FVIII during theincubation.

Because both the FVIII:Ag level and the FVIII:Chrom activity increasesupon incubation at buffer pH 8.1, suggesting a demasking effect of PEGrelease, the amount of FVIII applied to the gel accorded with themeasured FVIII:Chrom activity of the material without performing arelease reaction (100 mIU FVIII:Chrom for the anti-FVIII antibodies and300 mIU FVIII:Chrom for anti-PEG antibody)

As shown in FIG. 39, after 3 days incubation in buffer pH 8.1 nosubstantial changes could be found for the PEG-rFVIII conjugates but aslight blurring effect for both native rFVIII and the FVIII control withthe anti-FVIII immuoblots was observed. Longer incubation up to 7 daysresulted in degradation with non-detectable epitopes. By comparison ofthe different candidates, the Lys 40K br long and Lys 20K br longconjugates, showed the longest structural integrities. Some MW decreasein the heavy chain of the PEG-rFVIII conjugates occurred after 3 daysincubation as demonstrated by FIG. 40. After 7 days incubation in allconjugates the HC-A2 fragment appeared. A time-dependent decrease of themolecular weight of all domains was also observed with the anti-PEGimmunoblot (FIG. 41), which reflects some release of the bound PEG. Nosubstantial amounts of free PEG were detected. After 7 days incubationtime, all conjugates were degraded to such an extent that they cannot bevisualized by the FVIII-specific immunoblots (data not shown).

In vitro release of releasable PEG-rFVIII in a human FVIII-deficientplasma: To simulate the physiological conditions, in the second releaseexperiment the PEG-rFVIII conjugates with 20K and 40K PEG were incubatedin a human FVIII-deficient plasma at +37° C. The dissociation of the PEGwas investigated under these conditions by measuring the changes inFVIII chromogenic activity and FVIII antigen level. The PEG-rFVIIIconjugates together with a native rFVIII and the FVIII control werediluted to 0.1 μg/ml protein concentration and added to aFVIII-deficient plasma with an FVIII activity below 1% (George KingBio-Medical Overland Parks, Kans., USA) with 0.005% sodium-azide toprevent microbiological contamination during the incubation time.Samples were withdrawn at defined time points and tested immediately inthe case of FVIII chromogenic activity determination or aliquoted andfrozen at −80° C. for FVIII:Ag determination.

Because of a lack of material, PEG-rFVIII conjugates were resynthesizedfor this experiment, only the Lys 20K br short was from the firstproduction. The resynthesized Lys 20K br long had a lower PEGylationdegree. The resynthetized Lys 40K br long had also a lower PEgylationdegree, however due to different analytical methods, no directcomparison was feasible (Table 9). There were also some differences inthe specific activities, however they did not seem to correlate with thePEGylation degree (Table 11).

The specific activity of the native rFVIII decreased upon incubation inthe plasma system (FIG. 42). In contrast to this, the specific activityof the conjugates increased transiently upon incubation reaching amaximum level after approximately 10 hours. None of the conjugatesachieved the initial specific activity of the native rFVIII. There wereno substantial differences between the inactivation rates, determinedfrom the ascending part of the curves, between the different conjugatesand the native rFVIII (data not shown). The FVIII antigen level alsoincreased during the incubation (FIG. 43), which hint at a demaskingeffect. Similar to the FVIII activity results, none of the conjugatesachieved the initial level of the native rFVIII.

FVIII-deficient knockout mice were infused with either rFVIII orPEG-rFVIII in a target dose of 200 IU FVIII/kg bodyweight. Groups of 6mice per time point were used for each conjugate. To allow directcomparison of elimination curves independent of the FVIII dose applied,FVIII plasma levels were normalized relative to the FVIII concentrationfound in plasma 5 minutes after substance application (normalized %).

FIGS. 44-49 show plasma levels of FVIII after substance injection,either in IU FVIII/ml (Panels A) or normalized as a percent (Panels B).The exact amount of the injected material is shown in the appropriatefigure legends.

In general, all PEG-rFVIII showed improved pharmacokinetics over thenative control. The degrees of improvement of pharmacokinetic parameterswere calculated with statistical methods and are summarized in Table 10.

Immediately after infusion of PEG-rFVIII, the plasma FVIII activityincreased, reached a plateau between 3 hours and 6 hours with asubsequent decline. 24 hours after infusion, approximately 10 times morePEG-rFVIII activity was measured in the mouse plasma than afterinjection of native rFVIII.

Similar to the Lys 20K br short candidate, PEG-rFVIII 20K br longcirculated much longer than native rFVIII.

Recombinant FVIII, PEGylated with 40K br short PEG, circulated longerthan native rFVIII.

24 hours after injection, native rFVIII was close to the limit ofquantification, while PEG-rFVIII Lys 40K br long was still detectable 32hours after infusion.

The 60K conjugate with short releasable characteristics was eliminatedmuch slower than native rFVIII from mouse plasma.

The PEG-rFVIII Lys 60K br long conjugate circulated much longer inhemophilic mice than the native control rFVIII did. For directcomparison, the normalized elimination curves for all PEG-rFVIIIcandidates are summarized in FIG. 25. The control group is the mean ofall experiments, performed throughout the investigation (24 animals pertime point).

The results from the statistical evaluation are given in Table 18. Dataare given as increase in pharmacokinetic parameters versus the dedicatedcontrols, run together with each rFVIII conjugate. FVIII area under thecurve (AUC) was significantly increased for all candidates, but none ofthe candidates appears superior to the others. Increase in FVIIIhalf-life varied between the candidates. PEG-rFVIII Lys 20K br longresulted in a significant increase in FVIII half-life. For the 40Kcandidates, a statistical trend towards significantly increased FVIIIhalf-lives was observed, but a study extension with plasma samplingpoints up to 60 hours would be necessary to confirm significances. The60K variants gave no significant increase in half-lives. Mean residencetime was always higher for the PEG-rFVIII conjugates than for the nativecontrol. The highest increase in MRT was observed for the Lys 40K brshort candidate, whereas the Lys 60K conjugates gave the lowestincrease.

TABLE 18 Pharmacokinetic parameters for PEG-rFVIII (increase versuscontrol) FVIII AUC FVIII half life FVIII MRT rVWF Sample increase versuscontrol Lys 20K br short 3.6^(s) 1.4^(ns) 2.1 Lys 20K br long 4.1^(s)2.0^(s ) 2.6 Lys 40K br short 3.9^(s) 2.8^(s§) 3.9 Lys 40K br long4.8^(s) 2.0^(n§) 3.1 Lys 60K br short 4.7^(s) 0.9^(ns) 1.5 Lys 60K brlong 4.6^(s) 1.2^(ns) 1.9 ^(s)significant, ^(s§)statistical trend,^(ns)not significant data show the increase in pharmacokineticparameters versus the respective control group with native rFVIII, runtogether with each PEG-rFVIII conjugate.

Whereas in Table 18 results are given as relative increase versus theindividual control experiment, FIG. 51 shows the absolute data for AUCand half life from statistical evaluation together with the 95%confidence intervals to allow direct comparison between candidates.

The AUC for all PEG-rFVIII conjugates was clearly higher than for themean native rFVIII control. The 95% confidence intervals for thePEG-rFVIII did not overlap with those from the control. Within thePEG-rFVIII candidates the Lys 60K conjugates and the Lys 20K br longcandidate seemed to have the most pronounced effect. FVIII half-life wasalso increased with all PEG-rFVIII candidates.

Mean residence time was elevated for all PEG-rFVIII candidates versusthe native rFVIII control (range for native control was from 5.6 to 8.9hours). The highest MRT was found for the Lys 20K br long and the Lys40K br short and long candidates.

In summary, all PEG-rFVIII conjugates preserved the domain structurewithout any degradation. In contrast, all functional activities weredecreased, which could only be partially recovered upon in vitroincubation. However, it should be taken into account that the measuredvalues were influenced by the rate of three simultaneous reactions, i.e.the release of PEG moieties, the inactivation of PEG-rFVIII and theinactivation of liberated native rFVIII. The Lys 60K br long conjugatehad a higher specific activity and as a consequence an elevated thrombingeneration capacity. However, it did not show any other functional orstructural beneficial properties properties over the 20K or 40 KPEG-rFVIII conjugates in the tests performed.

FVIII-deficient mice were injected with rFVIII or PEG-rFVIII and FVIIIactivity in plasma was followed up to 32 hours. All PEG-rFVIIIconjugates showed slower elimination than that of the native rFVIIIcontrol. Whereas native rFVIII was eliminated in the mouse model in abiphasic manner with a faster initial phase and a slower terminal phase,the PEG-rFVIII candidates followed a more linear eliminationcharacteristic. The Lys 20K br short candidate resulted in an increasein the FVIII plasma levels up to 6 hours after injection into hemophilicmice, followed by a FVIII activity decline. The initial increase inFVIII activity might be explained by the release of releasable PEG fromthe PEG-rFVIII, thus recovering FVIII activity.

The slower elimination of FVIII activity with the PEG-rFVIII conjugatesmight also be a result of two overlapping effects, a longer circulatingPEG-rFVIII that continuously liberates native rFVIII, which is thencleared with the normal elimination rate.

The FVIII dose applied in the animal model was based on the detectableFVIII activity. Because specific activity of FVIII was lower for thePEG-rFVIII candidates, this resulted in 1.9- to 6.7-fold higher proteindoses for PEG-rFVIII conjugates than for the native rFVIII control.Slower elimination of PEG-rFVIII seems not to be dependent on the higherprotein dose applied, but further experiments are needed to assess theeffect of higher protein doses of native rFVIII on the FVIII plasmalevels, in comparison to similar protein doses of PEG-rFVIII.

1. A compound having the following structure:

wherein: POLY¹ is a first water-soluble polymer; POLY² is a secondwater-soluble polymer; X¹ is a first spacer moiety; X² is a secondspacer moiety; H_(α) is an ionizable hydrogen atom; R¹ is H or anorganic radical; R² is H or an organic radical; (a) is either zero orone; (b) is either zero or one; R^(e1), when present, is a firstelectron altering group; R^(e2), when present, is a second electronaltering group; Y¹ is O or S; Y² is O or S; and (vWF/F8) is a residue ofan amine-containing biologically active agent selected from the groupconsisting of a von Willebrand Factor moiety and a Factor VIII moiety.2. The compound of claim 1, wherein the amine-containing biologicallyactive agent is a von Willebrand Factor moiety.
 3. The compound of claim2, wherein the von Willebrand Factor moiety is human recombinant vonWillebrand Factor.
 4. The compound of claim 1, wherein theamine-containing biologically active agent is a Factor VIII moiety. 5.The compound of claim 4, wherein the Factor VIII moiety is humanrecombinant B-domain deleted Factor VIII.
 6. The compound of claim 5,wherein the Factor VIII moiety is human recombinant full length FactorVIII.
 7. The compound of claim 1, wherein the first water-solublepolymer is a poly(alkylene oxide) and the second water-soluble polymeris a poly(alkylene oxide).
 8. The compound of claim 1, wherein the firstwater-soluble polymer has a weight-average molecular weight of between10,000 Daltons to 85,000 Daltons and the second water-soluble polymerhas a weight-average molecular weight of between 10,000 Daltons to85,000 Daltons.
 9. The compound of claim 1, having a structure selectedfrom the group consisting of:

wherein, for each structure and in each instance, (n) is independentlyan integer from 4 to 1500, and (vWF/F8) is a residue of anamine-containing biologically active agent selected from the groupconsisting of a von Willebrand Factor moiety and a Factor VIII moiety.10. The compound of claim 1, having the following structure:

wherein, vWF is a residue of an amine-containing, recombinant human vonWillebrand Factor and (n), in each instance, is independently from 4 to1500.
 11. The compound of claim 1, having the following structure:

wherein, vWF is a residue of an amine-containing, recombinant human vonWillebrand Factor and (n), in each instance, is independently from 4 to1500.
 12. The compound of claim 1, having the structure

wherein, F8 is an amine-containing residue of a Factor VIII moiety and(n), in each instance, is independently from 4 to
 1500. 13. The compoundof claim 12, wherein the Factor VIII moiety is recombinant B-domaindeleted Factor VIII.
 14. The compound of claim 12, wherein the FactorVIII moiety is human recombinant full length Factor VIII.
 15. Thecompound of claim 1, having the following structure:

wherein, F8 is a residue of an amine-containing Factor VIII moiety and(n), in each instance, is independently from 4 to
 1500. 16. The compoundof claim 15, wherein the Factor VIII moiety is recombinant B-domaindeleted Factor VIII.
 17. The compound of claim 15, wherein the FactorVIII moiety is human recombinant full length Factor VIII.
 18. A methodcomprising contacting a polymeric reagent to an amine-containingbiologically active agent selected from the group consisting of a vonWillebrand Factor moiety and a Factor VIII moiety under conditionssuitable to form a covalent attachment between the polymeric reagent andthe biologically active agent, wherein the polymeric reagent has thefollowing structure:

wherein: POLY¹ is a first water-soluble polymer; POLY² is a secondwater-soluble polymer; X¹ is a first spacer moiety; X² is a secondspacer moiety; H_(α) is an ionizable hydrogen atom; R¹ is H or anorganic radical; R² is H or an organic radical; (a) is either zero orone; (b) is either zero or one; R^(e1), when present, is a firstelectron altering group; R^(e2), when present, is a second electronaltering group; and (FG) is a functional group capable of reacting withan amino group of an active agent to form a releasable linkage.
 19. Themethod of claim 18, wherein the releasable linkage is a carbamatelinkage.
 20. The method of claim 18, wherein the polymeric reagent has astructure selected from the group consisting of:

wherein, for each structure and in each instance, (n) is independentlyan integer from 4 to
 1500. 21. The method of claim 20, wherein theamine-containing biologically active agent is a von Willebrand Factormoiety.
 22. The method of claim 21, wherein the von Willebrand Factormoiety is human recombinant von Willebrand Factor.
 23. The method ofclaim 20, wherein the amine-containing biologically active agent is aFactor VIII moiety.
 24. The method of claim 23, wherein the Factor VIIImoiety is human recombinant B-domain deleted Factor VIII.
 25. The methodof claim 24, wherein the Factor VIII moiety is human recombinant fulllength Factor VIII.
 26. A composition comprising a compound of any oneof claims 1 though 17 and a pharmaceutically acceptable excipient.
 27. Amethod comprising administering a composition of claim 26 to a patient.28. A construct comprising a compound of claim 2 bound to at least oneFactor VIII moiety.
 29. The construct of claim 28, having an in vivohalf-life that is increased as compared to the in vivo half-life of aVWF molecule.
 30. The construct of claim 28, having an in vivo half-lifethat is increased as compared to the in vivo half-life of a Factor VIIImoiety not bound to said construct.
 31. The compound of claim 2, havingan in vivo half-life that is increased as compared to the in vivohalf-life of a VWF molecule.
 32. The compound of claim 31, wherein thein vivo half-life is increased by a factor of at least 1.5 as comparedto the in vivo half-life of a VWF molecule.
 33. The compound of claim31, wherein the in vivo half-life is increased by a factor of at least 2as compared to the in vivo half-life of a VWF molecule.
 34. The compoundof claim 4, having an in vivo half-life that is increased as compared tothe in vivo half-life of a FVIII molecule.
 35. The compound of claim 34,wherein the in vivo half-life is increased by a factor of at least 1.5as compared to the in vivo half-life of a FVIII molecule.
 36. Thecompound of claim 34, wherein the in vivo half-life is increased by afactor of at least 2 as compare to the in vivo half-life of a FVIIImolecule.